Fig. 2.
Clp-dependent loss of Mrf stability in high-zinc medium. (A and B) Accumulation of Mrf upon depletion of either ClpP1 in strain YL9 (A) or ClpP2 protease in strain YL10 (B). Cells were cultured in high-zinc Sauton’s medium and guide RNA corresponding to ClpP1 (A) or ClpP2 (B) was induced with Atc for 6 or 16 h prior to Mrf analysis. Uninduced cultures at corresponding time points and high- or low-zinc 96-h cultures of each strain were controls. The minor band below Mrf is unrelated to the FLAG signal that also appears in lysates of the parent strains. (C) Analysis of Mrf levels in YL2 (ctrl) strain or its isogenic ΔclpS mutant (YL15) at indicated time points of growth in low-zinc Sauton’s medium. (D) Coelution of Mrf from high- or low-zinc cultures of YL16—a derivative of YL9 that expressed ClpS6XHis—on Ni-NTA matrix. Mrf from high-zinc culture was analyzed upon depletion of ClpP1. The plots to the right of each immunoblot show the average normalized Mrf signal from three biologically independent experiments. *P < 0.05 and **P < 0.01 (t test).