All strains used in this work are described in Table 1. Streptococcus suis, S. dysgalactiae, S. pyogenes, Listeria and Enterococcus faecium strains were cultivated in brain heart infusion(BHI)broth, and the remaining strains in Luria-Bertani(LB)broth.
Table 1. Strains used in this study
The original plySs2 gene was chemically synthesized into pUC57 plasmid by Sangon Biotech(Shanghai, China). To assess the binding specificity of PlySs2, seven truncated fractions were constructed and genetically fused with an N-terminal enhanced green fluorescent protein(EGFP)cloned from pET-EGFP, generating the recombinant proteins EGFP-PlySs2, EGFP-PlySc, EGFP-PlySb, EGFP-62PlySb, EGFP-41PlySb, EGFP-35PlySb and EGFP-27PlySb, respectively. PlySs2 and its putative CD(PlySc)were additionally cloned and purified for examination of lytic activity. The primers used in this study are shown in Table 2. All gene fragments were cloned into pET28a(+)vector and transformed into E. coli BL21(DE3)cells for protein expression after verification via sequencing.
Table 2. Primers used in this study
Recombinant proteins were purified using His tag as described previously(Yang et al., 2012), with minor modifications. In brief, cells were cultured in LB containing 50 μg/mL kanamycin to OD600 of 0.4–0.6. After cooling to 16 ℃, expression was induced for 12 h via addition of isopropyl-β-D-thiogalactopyranoside(IPTG) to a final concentration of 0.2 mmol/ L. Cells were harvested and resuspended in buffer A(20 mmol/L Tris-HCl, 500 mmol/L NaCl, 20 mmol / L imidazole, pH 8.0) with 30% glyc erol. After soni cation, the supe rnatant was collected by centrifugation at 12, 000 g for 30 min at 4 ℃. Purification was achieved following the general protocol using a nickel nitrilotriacetic acid column, and subsequent washing and elution with imidazole solutions at concentrations of 40 and 400 mmol/ L, respectively. Collected fractions were dialyzed against buffer B(20 mmol/L Tris-HCl, pH 7.4)with 30% glycerol and then stored at -80 ℃ until use after quantitation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bradford assay.
Lytic activities of PlySc, PlySs2, EGFP-PlySc and EGFP-PlySs2 were confirmed using the turbidity reduction assay described previously(Gilmer et al., 2013), with minor modifications. Cells were washed once and resuspended in buffer B to a final OD600 of 1.0. Aliquots of 100 μL suspensions were mixed with PlySc, PlySs2, EGFP-PlySc or EGFP-PlySs2(each at a final concentration of 926 pmol/mL)in a 96-well plate, and changes in OD600 monitored immediately using a microplate reader (Synergy H1, BioTek, USA)at 37 ℃ for 60 min. Wells containing mixtures of bacterial cells and buffer B were used as controls. Lytic activity was calculated as initial OD600 divided by final OD600 in each well. All experiments were performed for three replicates.
To determine the binding activities of the EGFP-tagged fractions, mid-log phase cells were washed twice with phosphate-buffered saline(PBS, 137 mmol/L N aCl, 2.7 mmol/L KCl, 4.3 mmol/L Na2HPO4 · H2O, 1.4 mmol/L KH2PO4, pH 7.4) and mixed with 10 mmol/L EG FP, EGFP-PlySc, EGFP-PlySb, EGFP-62PlySb, EGFP-41PlySb, EGFP-35PlySb or EGFP-27Pl ySb at 37 ℃ for 30 min. Staining of EGFP-PlySs2 was performed in phosphate buffer with 750 mmol/L NaCl(PB, 750 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L Na2HPO4 · H2O, 1.4 mmol/L KH2PO4, pH 7.4)to suppress lytic activity. Subsequently, cells were washed twice with PBST buffer (PBS with 0.5% Tween-20) and resuspended in PBS, prior to fluorescence microscopy analysis(Delta Vision Personal DV, Applied Precision, USA).