Toxin Effectors of CDI and Rhs Systems Figure 7. The EC93 orphan region is transcribed. RNA from E. coli EC93, EC93 DcdiA-CTEC93DcdiIEC93, and EC93 DcdiA-CTo1EC93DcdiIo1EC93 was subjected to quantitative RT-PCR. The primer binding sites within the cdi locus are depicted schematically as arrows. The relative expression levels represent the mean 6 SEM for three independently isolated RNA samples. doi:10.1371/journal.pgen.1002217.g007 similarities to CDI systems, these observations suggest that Rhs proteins may be involved in intercellular competition. To test whether other Rhs systems encode toxin/immunity pairs, we examined rhs genes from D. dadantii 3937, which contains three full-length rhs genes that we have termed rhsA (Dda3937_01758), rhsB (Dda3937_02773) and rhsC (Dda3937_04312). Each of these rhs genes is closely followed by a small ORF that encodes a possible immunity protein. Additionally, the rhsC locus contains the two orphan rhs-CT gene fragments described above. We first tested RhsB-CT3937 for growth inhibitory function, because this domain Figure 8. The EC93 orphan region produces functional CdiIo1 immunity protein. EC93 expressing chimeric CdiAEC93-CTo1EC93 was used as an inhibitor strain in growth competition experiments. Inhibitor cells were co-cultured with wild-type EC93, EC93 deleted for the orphan rceegllisonco(mDpcdleiAm-CenTote1EdC9w3DitchdiaIo1pECla9s3m), iadn-bdorEnCe93coDpcydioAf-CcTdoiI1oE1CE9C39D3c(dcidIoi1IoE1C+9)3. 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.g008 contains an HNH endonuclease motif found in other cytotoxic proteins [21]. We expressed RhsB-CT3937 together with an ssrA(DAS)-tagged version of the putative RhsIB3937 immunity protein in E. coli cells, and found that growth was arrested upon degradation of RhsIB3937-(DAS) (Figure 10 and data not shown). The same results were obtained with the two orphan modules from D. dadantii 3937 (Figure 10 and data not shown), indicating that Rhs-CTo13937 and Rhs-CTo23937 also have growth inhibition activity. These results also demonstrate that the putative rhsI genes do in fact encode proteins with immunity function. Finally, we examined the specificity of RhsI-mediated immu- nity. Although each Rhs-CT/RhsI-(DAS) expression construct can be maintained stably in E. coli DsspB strains, these plasmids cannot be transformed into sspB+ cells due to the Rhs-CT toxicity that results from RhsI-(DAS) degradation (Figure 10 and data not shown). Therefore, we asked whether untagged RhsI protein could rescue cells from Rhs-CT toxicity and allow stable transformation of Rhs-CT/RhsI-(DAS) plasmids into sspB+ cells. Each of the three Rhs-CT/RhsI-(DAS) plasmids were introduced into sspB+ cells expressing individual RhsI proteins, and the transformed cell suspensions plated onto selective media containing L-arabinose to induce Rhs-CT/RhsI-(DAS) synthesis. Transformants carrying the Rhs-CT/RhsI-(DAS) expression plasmid were only obtained if the cells also expressed the cognate RhsI immunity protein (Figure 10). Therefore, each RhsI protein only confers immunity to its cognate Rhs-CT, demonstrating that the Rhs systems of D. dadantii 3937 encode polymorphic toxin/immunity pairs. Discussion The results presented here show that many CDI systems have a complex genetic organization in which the cdiBAI genes are PLoS Genetics | www.plosgenetics.org 8 August 2011 | Volume 7 | Issue 8 | e1002217