Figure 6.

CysK is required for CdiA-CT-mediated growth inhibition and tRNase activity in vivo. (A) The DAS-tagged CdiI immunity protein was targeted for proteolysis by SspB production (induced by L-arabinose at the indicated time point) to liberate either CdiA-CT or CdiA-CT(ΔI) proteins in cysK⁺ and ΔcysK cells. Cell growth was monitored by measuring the OD₆₀₀ as a function of time. (B) Cells from the experiment shown in A were removed at various times, and total RNA was isolated for Northern blot analysis. The gel migration positions of full-length and cleaved tRNA₂^Arg are indicated. (C) Complementation of the ΔcysK mutation. E. coli ΔcysK cells expressing the CdiA-CT/CdiI-DAS complex were transformed with plasmids that produce CysK, CysK(K42A), or CysM under control of the arabinose-inducible P_BAD promoter. Transformants were selected on LB agar plates supplemented with 0.2% L-arabinose and the appropriate antibiotics. Transformation of E. coli ΔcysK ΔsspB cells serves as a control to demonstrate that CdiI-DAS degradation is required for growth inhibition.