FIG 2.

Zinc specifically inhibits SpeB activity at a posttranslational level, and a zinc chelator reverses this process. (A) Concentrated supernatant samples from 5448WT grown in the absence of metals and/or chelator were mixed with 200 μM (final concentration) each of the metals CaCl₂, MgCl₂, MnCl₂, and ZnSO₄ in the presence or absence of DTPA. Cysteine protease activity of SpeB was detected using a fluorescent-enzyme assay as described in Materials and Methods. The data presented are means and SD (n = 4; experiments were repeated four times in triplicate), and each data point represents the percentage of maximum protease activity at 200 μM the specified metal and/or DTPA. The data were normalized to the total protease activity obtained in the absence of metals or chelator (control group). Two-way ANOVA analyses revealed significant interaction (P < 0.001) between bacterial cultures containing different metal groups and their DTPA treatments. a indicates a significant difference (P < 0.001) within treatments (with and without DTPA) and between cultures containing different metal groups and the specified group by Bonferroni post hoc analysis. (B) Analysis of protease activity in the presence of increasing concentrations of ZnSO₄. The data are means ± standard errors (SE) (n = 4; experiments were repeated four times in triplicate). Ligand binding and a sigmoidal dose-response regression curve fit were used to fit the line (R² = 0.9950; P < 0.001), and we obtained an IC₅₀ of 27.29 ± 1.08 μM (P < 0.001) for zinc-mediated SpeB inhibition. (C) DTPA can reverse the protease inhibition of SpeB by chelating ZnSO₄ (200 μM) when added in increasing concentrations. The data are means and SD (n = 3; experiments were repeated three times in triplicate), and two-way ANOVA revealed an interaction (P < 0.001) between zinc and DTPA. a indicates a significant difference (P < 0.001) within treatments (increasing DTPA concentrations) and between the zinc-treated and control groups, and b indicates a significant difference (P < 0.001) between 200 μM DTPA-treated culture and no treatment (No DTPA) within the zinc group (Bonferroni post hoc analyses).