Abstract
BackgroundEndolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33).ResultsBioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins.ConclusionsPlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.
Highlights
Endolysins produced by bacteriophages lyse bacteria, and are considered a novel type of antimicrobial agent
Pfam and CDD analysis showed that PlyBt33 was composed of two functional domains (Figure 1b), the Nterminal catalytic domain and the C-terminal cell wall binding domain
According to homology-based endolysin classification [1], PlyBt33 is a putative member of the N-acetylmuramoyl-L-alanine amidases
Summary
Endolysins produced by bacteriophages lyse bacteria, and are considered a novel type of antimicrobial agent. 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 [5,6,7,8,9,10]. The endolysin C-terminus 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]
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