5 M NaCl, pH 8.0), and then ultrasonic treatment was performed on ice. The supernatant was collected by centrifugation, and the elution buffer (20 mM Na3PO4, 0.5 M NaCl, 0.5 M imidazole, pH 8.0) in accordance with 1:20

were added. The protein of interest (VirB1-89KCHAP) was purified on His GraviTrap column prepacked with Ni Sepharose 6 Fast Flow, then IWR-1 in vitro washed with binding buffer until the absorbance reaches the baseline. The target protein was eluted with elution buffer using a linear gradient. The elution was checked by SDS-PAGE (12%) and fractions containing the interest protein were further purified by gel filtration chromatography using Superdex-75 column. Peak elution fractions were analyzed by gel electrophoresis and those containing pure protein were pooled and concentrated in an Amicon apparatus (Millipore) with a 10-kDa molecular weight cutoff membrane, then stored in 0.1-ml aliquots at −80°C. The protein concentration was determined by using the Pierce BCA protein assay kit. Determination of the lytic activity of VirB1-89KCHAP To determine the peptidoglycan-degrading activity of VirB1-89KCHAP, zymogram analysis was performed as described previously [32, 33]. Peptidoglycan isolated and purified from S. suis 2 was added into 12% polyacrylamide gels to a final concentration of 100 mg/ml [24, 34]. After Hydroxychloroquine cost electrophoresis,

the gels were incubated at 37°C in renaturation buffer (20 mM sodium phosphate buffer, 0.1% Trition X-100, 10 mM MgCl2, pH 8.0) for 16 h, and then stained with 1% methylene blue containing 0.1% KOH. The deionized water was used for depolarization. The bacteriostatic activity of VirB1-89KCHAP was determined Histamine H2 receptor with slip-agar diffusion method [35]. A small piece of filter paper loaded with purified VirB1-89KCHAP was placed on a 1.5% agar plate inoculated with S. suis 2 cells, and then bacteriostatic rings of protein-sensitive slips were generally observed

after incubation and the diameters of bacteriostatic rings were measured with a vernier caliper. Hen egg white lysozyme and BSA were used as positive and negative controls, respectively. The effect of pH and temperature on the enzymatic activity of VirB1-89KCHAP The effect of pH and temperature on the enzymatic activity of VirB1-89KCHAP was determined as previously described with minor modifications [31]. Purified VirB1-89KCHAP protein was added to 200 μl the dried cells of M. lysodeikticus as substrate. To determine the optimal pH value, the enzyme activity was monitored at 37°C with different pH values ranging from 3.0 to 11.0. The optimum temperature of the enzyme was tested at the temperature ranging from 20°C to 70°C at the optimum pH value. For the thermal stability estimation, the enzyme was pre-incubated at temperatures between 30°C and 90°C for 30 min, and the remaining activity was determined under the optimum reaction conditions. In vivo virulence studies To determine whether the virB1-89K gene is necessary for the virulence of the highly pathogenic S.