Endolysins are enzymes produced by bacteriophages (phages) at the end of their life cycles to lyse the cell walls of host cells and release mature progeny phage particles [1, 2]. Most endolysins require a second phage protein, holin, to create pores in the cytomembrane and enable them to pass through to reach their substrate, a cell wall peptidoglycan [3, 4]. Because of their potential as novel antibacterial agents, the characteristics of several endolysins have previously been studied [5–10].
Endolysins of phages isolated from Gram-positive bacteria typically contain two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain . The catalytic domain belongs to one of the four families of peptidoglycan hydrolases, which are classified according to catalytic site-specificity: N-acetylglucosaminidases, N-acetylmuramidases (lysozymes), N-acetylmuramoyl-L-alanine amidases, and endopeptidases [1, 11]. By contrast, the cell wall binding domain is divergent and can distinguish discrete cell wall epitopes. Usually, one cell wall binding domain determines the endolysin strain specificity [11, 12]; however, there are sometimes more than one [7, 13, 14] or even no cell wall binding domains [15, 16]. The endolysin C-terminus nevertheless sometimes appears to be essential for catalytic activity, as several reports showed that the enzymatic activity is abolished after removal of the C-terminus [17, 18].
Bacillus thuringiensis belongs to the Bacillus cereus group, which includes two very closely related species: B. cereus and Bacillus anthracis. B. thuringiensis is an insect pathogen that forms an insecticidal crystal protein during sporulation . B. anthracis is the anthrax pathogen, while B. cereus is a food contaminant . Because of the multidrug resistance of B. anthracis[21, 22], several of its phage or prophage endolysins have been expressed, purified, and characterized. There have also been some attempts to use these endolysins to cure the disease caused by B. anthracis[8, 9, 11, 17, 18, 23]. Practical applications of endolysins were enabled by studies on functional domain composition, optimal reaction conditions, and species- or strain-specificity. For example, combining the catalytic domain of one endolysin with the cell wall binding domain of another changed the specificity or activity .
Until now, only two bacterial cell wall hydrolases from B. thuringiensis phage GIL01 have been reported , and little is known about their functional domain composition. The lytic activity of one of these hydrolases was limited to B. thuringiensis israelensis, while the other exhibited a broader cleavage spectrum in lysing two other Gram-positive species, B. subtilis and Micrococcus lysodeikticus.
Phage BtCS33 is a Siphoviridae family member that was isolated from B. thuringiensis kurstaki strain CS-33 . The BtCS33 genome has been sequenced and a potential endolysin gene, orf18, was identified using bioinformatics. The gene product was named PlyBt33. In this study, we analyzed the functional domain composition of PlyBt33 using bioinformatics, and then demonstrated its biological activity after separately expressing the catalytic and cell wall binding domains in Escherichia coli. PlyBt33 showed a broad lytic spectrum against the tested Bacillus strains. Additionally, its cell wall binding domain exhibited low amino acid sequence similarity to previously reported domains.