A Novel Small Acid Soluble Protein Variant Is Important for Spore Resistance of Most Clostridium perfringens Food Poisoning Isolates
Figure 6
DNA binding properties of purified recombinant His6-tagged rSsp4.
Panel A, purity and stability of Coomassie blue (top panel) or His6-tag Western blotted, purified His6-tagged rSsp4 proteins used in Fig. 6 B and C DNA binding experiments. Panel B, EMSA analysis of purified F4969, SM101 and 01E809 rSsp4 binding to C. perfringens DNA. Lane 1, free biotin-labeled C. perfringens DNA; lanes 2–4, indicated amounts of purified F4969 rSsp4 incubated with C. perfringens biotin-labeled DNA; lanes 5–7, indicated amounts of purified SM101 rSsp4 incubated with C. perfringens biotin-labeled DNA; and lanes 8–10, indicated amounts of purified 01E809 rSsp4 incubated with C. perfringens biotin-labeled DNA. Panel C, effects of NaCl on binding of rSsp4 to DNA. Beads (100 µg) containing calf thymus DNA (Sigma) were incubated with 100 ng of rSsp4 from the indicated strain. After washing with 0, 0.25 M, 0.50 M, 0.75 M or 1.0 M NaCl, the beads were boiled and then analyzed by SDS-PAGE. Lane 1, input protein; lane 2, 100 µg DNA-free beads incubated with 100 ng purified rSsp4 from SM101, 01E809 or F4969; lanes 3–7, 100 µg DNA-containing beads incubated with 100 ng purified rSsp4 from SM101, 01E809 or F4969 and washed with indicated concentrations of NaCl.