Figure 2.
Effect of LdCsm1 mutations on ATP binding and ATP-stimulated ssDNA cleavage activity by LdCsm effector. (A) ATP binding assay with LdCsm1-mutated effectors. Red arrowheads indicate the Csm-ATP complex. (B) Reporter DNA assay on the ssDNase activity of LdCsm1-mutated effectors. The ssDNase activity of each LdCsm effector in the absence of ATP was set to 100% to which the relative activity was calculated for the same effector in the presence of 100 μM ATP. (C) RNA-activated ssDNA cleavage assay with the mutated LdCsm effectors. Incubation time was for 30 min except for Csm1P2D543A and Csm1P2DxD (120 min). (D) Anti-plasmid activity by the LdCsm system and LdCsm1 D541 mutants. Electroporated E. coli cells were plated on nutrient media containing either glucose or arabinose as the carbon source overnight at 37°C. Concentrations of the L-arabinose inducer are indicated on the top of the panel, with glucose (0.5% Glu) as a reference.