Toxin Effectors of CDI and Rhs Systems Figure 6. The EC93 orphan cdiA-CT/cdiI module is functional in contact-dependent growth inhibition (CDI). A) The wild-type EC93 and orphan chimera CDI systems are shown schematically. The cdiA-CTEC93/cdiIEC93 region was deleted and orphan module fused onto the cdiAEC93 gene at the VENN encoding sequence. B) Growth competitions. CDI+ inhibitor cells were co-cultured with target cells expressing either CdiIEC93 or orphan CdiIo1EC93 immunity proteins. Viable target cells were quantified by plating on selective media to determine the number of colony forming units (cfu) per milliliter. doi:10.1371/journal.pgen.1002217.g006 peptide motif (PxxxxDPxGL) analogous to the VENN motif in CdiA proteins [12,19]. The parallels between CDI and Rhs systems extend to their genetic organization, with many rhs loci containing numerous ‘‘silent cassettes’’ that resemble CDI orphan modules [12]. Rhs systems also appear to undergo complex rearrangements that diversify Rhs-CT sequences. For example, in one Rhs region shared by Y. pseudotuberculosis strains IP31758 and IP32953, the rhs-CT and putative rhsI sequences are completely unrelated to one another, but the surrounding genomic regions are clearly homologous between the strains (Figure 9). This homology includes rhs coding sequence upstream of the region encoding DPxGL and nearly identical rhs-CT/rhsI orphan modules downstream of the rhs genes (Figure 9 and Figure S8). In addition, orphan rhs-CT/rhsI modules from a given species are often found fused to full-length rhs genes in other bacteria. For example, one of the rhs loci in D. dadantii 3937 contains two orphan rhs-CT/rhsI modules. Both of these predicted orphan Rhs-CT3937 proteins are related to the C-terminal regions of full-length Rhs proteins: Rhs- CTo13937 (E0SGM0) is related to the CT region of a putative Rhs repeat protein (C3K5K6; 49% identity in 147 residues following the DPxGL) from Pseudomonas fluorescens SBW25, and Rhs- CTo23937 (E0SGM2) is related to the CT of a predicted YD- repeat protein (C6CNW6; 99% identity in 116 residues) from Dickeya zeae 1591. In general, the functions of Rhs proteins are unknown, but data from Hill and colleagues suggest that that the E. coli rhsA locus may encode a toxin/immunity pair [20]. In conjunction with the Figure 5. CdiA-CTo1EC93 inhibits the growth of E. coli cells. A) Growth curves of E. coli DsspB cells expressing CdiA-CTo1EC93/CdiIo1EC93- DAS. Degradation of CdiIo1EC93-DAS was initiated by the addition of L- arabinose to induce SspB synthesis. Control cells express SspB(D47), which does not deliver CdiIo1EC93-DAS to the ClpXP protease. Growth curves with square symbols represent control strains expressing SspB or SspB(D47), but not CdiA-CTo1EC93/CdiIo1EC93-DAS. B) Analysis of in vivo CdiA-CTo1EC93 tRNase activity. Total RNA was isolated from cells expressing CdiA-CTo1EC93/CdiIo1EC93-DAS at varying times after L- arabinose induction. Samples were run on polyacrylamide gels followed by staining with ethidium bromide (EtBr) or Northern blot analysis using probes specific for tRNA1BAla and tRNAHis. doi:10.1371/journal.pgen.1002217.g005 PLoS Genetics | www.plosgenetics.org 7 August 2011 | Volume 7 | Issue 8 | e1002217