Figure 4.

CdiA-CT^Ykris cleaves bacterial RNAs. (A) CdiA-CT^Ykris RNase activity in vitro. Purified CdiA-CT^Ykris variants were incubated with total E. coli RNA as described in the Methods. Reactions were quenched at the indicated times and run on denaturing polyacrylamide gels and visualized by staining with ethidium bromide. Where indicated (+CdiI), reactions were supplemented with purified CdiI^Ykris immunity protein. (B) CdiA-CT^Ykris RNase activity in vivo. The indicated CdiA-CT^Ykris variants were activated inside E. coli cells, and total RNA isolated for denaturing polyacrylamide gel analysis. (C) Competition co-culture assays. Inhibitor cells expressing the indicated CdiA-CT^Ykris toxins were co-cultured at a 1:1 ratio with target cells that either lack (–) or carry (+) the cdiI^Ykris immunity gene. The competitive index is the ratio of target to inhibitor cells at 3 h divided by the initial inhibitor to target cell ratio. Data for three independent experiments are presented ± the standard error of the mean.