(a), (b), (c), and (d). Filter papers were soaked in the crude extract suspended in 20 mM Tris-HCl (pH8.0) of PlyBt33 (a), PlyBt33-N (b), and PlyBt33-IC (c) from E. coli M15, and E. coli M15

containing pQE-30 (d), and placed onto the bacterial lawn of B. thuringiensis HD-73. (e) Lysis of viable cells using purified PlyBt33 and PlyBt33-N. Tests were performed in 20 mM Tris-HCl with a final protein concentration of 2 μM at 37°C. Crude extract of E. coli M15 containing pQE-30 was used as a control to treat B. thuringiensis strain HD-73. Figure 5 Characterization of the endolysin PlyBt33. (a) Lysis of viable cells from five different Bacillus species and one E. coli strain by PlyBt33. Tests were carried out with a final protein concentration of 2 μM at 37°C in 20 mM Tris-HCl (pH 8.0). The initial OD600 of each strain suspension was 0.8. Crude extract of E. coli M15 containing pQE-30 was used as a control to treat B. thuringiensis Foretinib solubility dmso strain HD-73. (b) pH-dependent activity of PlyBt33. Tests were carried out with a final protein concentration of 2 μM at 37°C in 20 mM Tris at varying pH levels. (c) Temperature-dependent

activity of PlyBt33. Tests were carried out with a final protein Salubrinal cell line concentration of 2 μM in 20 mM Tris-HCl (pH 8.0) at varying temperatures. (d) Temperature stability of PlyBt33. Proteins were first treated at different temperatures for 1 h and then the tests were carried out with a final protein concentration of 2 μM at 37°C in 20 mM Tris-HCl (pH 8.0). In (b), (c), and (d), decrease of OD600 (%) = (1− the absorbance of the bacterial suspension at the end of each treatment / the absorbance at the beginning of each treatment) × 100%. The effects of pH and temperature on PlyBt33 lytic activity were investigated. Lytic activity against the tested strains was observed in the pH range of 7.0–12.0, with an optimal pH of 9.0 (Figure 5b). The optimum reaction temperature was 50°C (Figure 5c), and lytic activity gradually decreased as temperature increased from 30–60°C (Figure 5d). Following treatments at 40°C and 60°C for 1 h, lytic activity was reduced by 40% and 60%, respectively. Cell wall binding activity

of PlyBt33-IC According to previous reports, the C-termini of several characterized Gram-positive endolysins comprised one or several second SH3 family cell wall binding domains [11, 14, 30]. Pfam analysis of PlyBt33 showed that the PlyBt33 C-terminus consisted of an Amidase02_C domain, which was present in several endolysins [9, 18]. We aligned the PlyBt33 C-terminus with other characterized cell wall binding domains from Bacillus phage or prophage endolysins, and observed limited similarity. However, the highest similarity was found with the C-termini of PlyG, PlyL, PlyBa04, and PlyPH (Figure 1). Kikkawa et al. previously reported that amino acid residues L190 and Q199 of endolysin PlyG were critical for the cell wall binding activity of PlyG to B. anthracis[32].