Toxin Effectors of CDI and Rhs Systems Figure 3. Interchange between cdiA and orphan cdiA-CTs. A) ACT comparison of the CDI systems (region I) from Y. pestis CO92 (top) and Y. pestis Microtus 91001 (bottom). The cdi loci lie within a highly conserved genomic region (red blocks denote nucleotide conservation), but the sequences encoding CdiA-CT diverge beginning at the VENN region. A 3,557 bp deletion in Y. pestis Microtus 91001 has fused the orphan cdiA-CT/cdiI module to the full-length cdiA gene (highlighted in yellow). B) ACT comparison of region II cdi loci from Y. pseudotuberculosis strains IP31758 (top) and PB1/+ (bottom). The cdi loci lie within a highly conserved genomic region (red denotes nucleotide conservation), but the cdiA-CT sequences exhibit complex rearrangements between the two strains. The orphan cdiA-CTo4PB1(II)/cdiIo4PB1(II) module of strain PB1/+ is essentially identical to the cdiA-CTIP31758(II) and cdiIIP31758(II) sequences from strain IP31758 (99.1% identity over 3,648 nucleotides; highlighted in yellow). Additionally, the orphan cdiA-CTo1PB1(II)/cdiIo1PB1(II) module is nearly identical to the orphan cdiA-CTo2IP31758(II)/cdiIo2IP31758(II) module (98% identity over 2,656 nucleotides, highlighted in blue), and the cdiA-CTo2PB1(II)/cdiIo2PB1(II) module is nearly identical to a cdiA fragment (and associated cdiI gene) found between the two orphan modules in strain IP31758 (98% identity over 1,073 nucleotides; highlighted in green). doi:10.1371/journal.pgen.1002217.g003 2 hours (Figure 5A). In contrast, growth continued unabated upon induction of SspB(D47) (Figure 5A), which lacks the C-terminal motif required for binding to ClpXP. Analysis of total cellular RNA revealed cleavage of tRNAs in the cells expressing wild-type SspB, but not in those expressing SspB(D47) (Figure 5B). These results demonstrate that CdiA-CTo1EC93 activity is unmasked upon CdiIo1EC93 degradation. Additionally, the temporal correla- tion between growth arrest and tRNA cleavage strongly suggests that the tRNase activity of CdiA-CTo1EC93 is responsible for growth inhibition. To test whether other orphan cdiA-CT/cdiI modules have growth inhibition activity, we examined orphan gene pairs from Dickeya dadantii 3937 and E. coli EC869. D. dadantii 3937 contains one orphan cdiA-CT/cdiI module in the region I cdi locus. We cloned this module and added a C-terminal His6 epitope tag onto the predicted CdiIo13937 protein. Overproduced CdiA-CTo13937 protein co-purified with CdiIo13937-His6 during Ni2+-affinity chromatography, indicating a binding interaction between these proteins (Figure S6A). Moreover, CdiA-CTo13937 inhibited the growth of E. coli cells upon degradation of ssrA(DAS)-tagged CdiIo13937 (Figure S6B). Examination of the orphan cdiA-CTo11/ cdiIo11 module from E. coli EC869 gave similar results, except growth inhibition was delayed compared to the D. dadantii 3937 orphan system (Figure S6). These data demonstrate that other orphan cdiA-CT/cdiI modules also encode functional toxin/ immunity pairs. The EC93 orphan cdiA-CTo1EC93 lacks translation initiation signals, suggesting the encoded protein is not synthesized under normal conditions. By analogy with other two-partner secretion proteins, full-length CdiA proteins are secreted through the inner membrane via the general secretory pathway and assembled onto the cell surface through interactions with CdiB [4,18]. The EC93 orphan cdiA-CTo1EC93 gene also lacks a signal sequence and thus would not be delivered to the cell surface if it were expressed. Therefore to test whether CdiA-CTo1EC93 and CdiIo1EC93 are functional in the context of cell-mediated CDI, we replaced the EC93 cdiA-CTEC93/cdiIEC93 region with the corresponding se- quences from the EC93 orphan module. The resulting construct PLoS Genetics | www.plosgenetics.org 5 August 2011 | Volume 7 | Issue 8 | e1002217