FIG. 4.

The role of divalent ions in the cytotoxic activity from S. marcescens culture filtrates. (A and C) The relative cytotoxicity of each sample was determined versus control cells treated with an equal volume of PBS, as described in the legend to Fig. 1. Culture filtrates prepared from S. marcescens MB1911 (A) or multiple strains of different origins (C) were treated at 4°C for 24 h and then at 37°C for 1 h with either 50 mM EDTA or 1,10-phenanthroline or in the absence of additional reagents (untreated). Excess protease inhibitors were removed by dialysis into PBS. Serial dilutions of the dialyzed filtrates were assayed. (B) Culture filtrates from S. marcescens MB1911 were prepared as described for panel A and then incubated with 2.1 mM Zn²⁺ for 1 h at 22°C prior to application to HeLa cell monolayers. The data from three separate experiments performed in replicates of at least six were averaged. The error bars indicate standard deviations. Statistical significance is as follows: (A) ∗, P = 0.007, and ∗∗, P < 0.0001; (B) P < 0.0001 for the 1,10-phenanthroline-treated metalloprotease compared to the sample with the zinc addback; (C) ∗, P < 0.0001, and **, P = 0.025 compared to the untreated culture filtrates.