Validation of proteomic hits reveals that WhiB1 and CarD are likely Clp protease substrates.

(A) Quantitative PCR to determine transcript levels of over-represented proteins upon depletion of Clp protease using RNA generated from Mtb P750-clpP1P2DAS after growth for 48 h in the presence or absence of ATc (1.5 µg/mL). Relative standard curves were generated for each probe set, and sigA transcript was used as an endogenous control. Data are represented as mean fold change, normalized to transcript in (−) ATc cultures +/− SEM of technical replicates. Protein ratios are derived from TMT experiments described in Figure 2, and represented as average ratios +/− standard deviation of biological replicates (B) Fluorescence (485/538) was measured for N- and C-terminal GFP fusions constructed for WhiB1 (left) and CarD (right), and induced for 8 hours in wildtype or clpP2-ID Msm with ATc (100 ng/mL). In clpP2-ID, ATc simultaneously induced fusion protein production and degradation of ClpP2. (C) Fluorescence (485/538) was measured for GFP fusions bearing a variable number of C-terminal residues from WhiB1 in clpP2-ID Msm, grown in the absence or presence of ATc (100 ng/mL) for 8 hours. In (B) and (C), data are represented as mean RFU +/− standard deviation of biological replicates. Asterisks denote a p-value <0.05 determined by t-test.