Figure 5.

Re-programming the Mtb Csm system for cA₄-responsive immunity. (A) In vitro activity of TsuCsx1. The reaction contained 0.5 μM TsuCsx1 dimer, 100 nM 5′-³²P-labelled RNA A1, 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 and was conducted at 35°C for 1, 5, 15, 30 min. Activators were added as indicated; Csm-derived cOAs were from a 2 h reaction, otherwise as described for Figure 3. TsuCsx1 was activated by cA₄ but not cA₆, and Csm-derived cOAs are able to induce Csx1 ribonuclease activity in vitro. (B) Plasmid immunity assay using pUC19 lacZα-targeting Csm effector complex in Escherichia coli C43 (pCsm1–5_Csm6/tsuCsx1 and pCRISPR, Supplementary Figure S4). The ribonuclease was either the cognate Csm6 or TsuCsx1. Cells were transformed with pRAT (control plasmid) or pRAT-Target (target plasmid) and 10-fold serial dilutions were plated on selective plates containing arabinose for induction of target transcription. TsuCsx1 confers the same level of plasmid immunity as the cognate Mtb Csm6.