The PlyBt33 C-terminus was expressed, purified, and labeled with fluorescein isothiocyanate (FITC). After mixing FITC-PlyBt33-IC with the bacterial suspension for 5 min, the cells were visualized under a fluorescence microscope, and binding between FITC-PlyBt33-IC and the surface of B. thuringiensis HD-73 was apparent (Figure 6a). The

binding ability assay was also repeated with a higher FITC-PlyBt33-IC concentration find more (0.05 mg/ml). At this concentration, homogenous binding of FITC-PlyBt33-IC to the cell surface was observed (data not shown), in contrast to the random binding pattern seen at the lower concentration. FITC-labeled bovine serum albumin (BSA) showed no binding to HD-73 (Figure 6b), and the HD-73 cell suspensions used as a control showed no fluorescence (Figure 6c). FITC-PlyBt33-IC also bound to B. subtilis 168, while no binding was detected in E. coli (data not shown). The binding activity of PlyBt33-IC was consistent Selleckchem Luminespib with its lytic specificity. Figure 6 Binding ability of FITC-PlyBt33-IC to viable cells of B. thuringiensis HD-73, as observed by phase contrast (upper panels)

and fluorescence (lower panels) microscopy. (a) Binding of FITC-PlyBt33-IC to the entire surface of HD-73; (b) No binding of FITC-BSA to HD-73 was observed; (c) HD-73 cell suspension with no protein was used as a control. Discussion In the present work, we expressed and determined the activity of endolysin PlyBt33 from B. thuringiensis phage BtCS33. The endolysin was found to be a putative N-acetylmuramoyl-L-alanine

amidase, and was composed of an N-terminal catalytic domain and a C-terminal cell wall binding domain. PlyBt33 maintained 40% of its lytic activity against bacterial cells following treatment at 60°C for 1 h. Though PlyBt33 exhibited a high sequence similarity (67%) to endolysin PlyPH, their characteristics were quite different. PlyPH was a B. anthracis putative prophage origin endolysin that could lyse B. anthracis and B. cereus, and had a broad optimal pH range (pH 4.0–10.5) [9]. By contrast, PlyBt33 exhibited lytic activity between pH 7.0–12.0, with an optimal pH of 9.0. The differences DOCK10 between the amino acid sequences of these two endolysins may cause differences in pI (putative pI 8.51 for PlyBt33 and 6.15 for PlyPH) and different surface net charges. Low et al.[23] reported that the net charge of endolysin PlyBa04 influenced its lytic activity and specificity, which might explain the different pH ranges of these two endolysins. Moreover, the lytic spectrums of PlyBt33 and PlyPH were also different. PlyBt33 could hydrolyze all tested Bacillus strains from five different species, while PlyPH could only lyse B. anthracis and B. cereus. Alignments of the putative cell wall binding domains of PlyBt33 and PlyPH revealed a low similarity (about 20%).