In the presence of agents such as EDTA, which permeabilize the outer cell membrane [29], LysB4 could be successfully applied exogenously to control

Gram-negative bacteria as well as Gram-positive bacterial pathogens. Methods Bacterial strains, phage and growth conditions B. cereus ATCC 10876 was used as the host for bacteriophage B4 (KCTC 12013BP) and the substrate for the LysB4 endolysin. E. coli BL21 (DE3) was used as the host for expression of the AZD5363 cost recombinant LysB4. Bacterial strains that were used for antimicrobial spectrum determination are described in Table 2 along with the results. All the bacterial strains were routinely grown at 37°C in Luria-Bertani (LB) broth medium (Difco). Ampicillin (50 μg/ml) was added when necessary. Bafilomycin A1 Cloning, expression, and purification of LysB4 The endolysin gene (lysB4) was amplified from the genomic DNA of the bacteriophage B4 by GSK872 ic50 polymerase chain reaction (PCR) using primers lysB4F (5′-AGTGGAAGTCATATGGCAATGGCATTA-3′) and lysB4R (5′-TAAAAAAAGGATCCCCGAAGGACTTCC). The PCR product was cloned into pET15b (Novagen), which has an N-terminal hexahistidine (His)-tag sequence. The correctly cloned plasmid was transformed into competent E. coli BL21 (DE3). Expression of the recombinant LysB4 was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside at OD600

1.0, followed by incubation for an additional 5 h at 30°C. Bacterial cells were suspended in lysis buffer (50 mM potassium phosphate, 200 mM sodium chloride, pH 7.0) and disrupted by sonication (Branson Ultrasonics). After centrifugation at 15,000 × g for 20 min, the supernatant was passed through a Ni-NTA Superflow column (Qiagen), and purification of the recombinant LysB4 was performed according to the manufacturer’s instructions. Thymidylate synthase The purified protein was stored at -80°C until use after the buffer was changed to the storage buffer (50 mM potassium

phosphate, pH 8.0, 200 mM NaCl, 30% glycerol) using PD Miditrap G-25 (GE Healthcare). Lytic activity assay The lytic activity of the endolysin against bacterial cells was assayed by monitoring the decrease in OD600 [30]. B. cereus ATCC 10876 or other bacteria were cultivated to exponential phase. Cells were harvested and resuspended with the reaction buffer (50 mM Tris-HCl, pH 8.0) to adjust OD600 to 0.8-1.0. When needed, 0.1 M EDTA was used to treat the Gram-negative bacteria after harvesting, as described previously [31]. The endolysin (100 μl) was added to the cell suspension (900 μl) followed by incubation at room temperature, unless indicated otherwise. OD600 values were monitored over time. The lytic activity was calculated after 5 min as followed; ΔOD600 test (endolysin added) – ΔOD600 control (buffer only)/initial OD600. To evaluate the effect of pH on LysB4 enzymatic activity, the endolysin (5 μg) was added to B. cereus cells suspended with a variety of buffers: 0.