Toxin Effectors of CDI and Rhs Systems Figure 4. The tRNase activity of CdiA-CTo1EC93 is blocked by the binding of CdiIo1EC93. A) Analysis of CdiA-CT/CdiI binding. Purified CdiA- CT and CdiI-His6 proteins were mixed at equimolar ratios then purified by Ni2+-affinity chromatography. Input samples represent the protein mixtures prior to chromatography. Unbound fractions contain proteins that failed to bind the affinity resin. Bound proteins were eluted from the affinity resin with imidazole. All fractions were analyzed by SDS-PAGE. B) Northern blot analysis of CdiA-CTUPEC536 and CdiA-CTo1EC93 tRNase activity. S100 fractions containing cellular tRNA was treated with purified CdiA-CT and/or CdiI-His6 proteins and then analyzed by Northern blot hybridization using probes specific for tRNA1BAla and tRNAHis. doi:10.1371/journal.pgen.1002217.g004 produces a chimeric molecule in which the CdiA-CTo1EC93 is fused to CdiAEC93 at the VENN motif (Figure 6A). E. coli expressing the CdiAEC93-CTo1EC93 chimera inhibited the growth of target cells expressing the CdiIEC93 immunity protein ,105-fold compared to control CDI2 inhibitor cells, but target cells expressing the orphan CdiIo1EC93 were completely protected from growth inhibition (Figure 6B). In contrast, CdiIo1EC93 was unable to protect target cells from growth inhibition mediated by CdiAEC93 (Figure 6B). Thus, the EC93 orphan CdiA-CTo1 is functional in contact-dependent growth inhibition when it is part of a full-length CdiA protein. The Orphan CdiI Immunity Protein Is Expressed in EC93 To determine whether orphan cdiA-CT/cdiI modules are expressed, we isolated total RNA from E. coli EC93 and performed quantitative RT-PCR using primer pairs to amplify from potential cdiA-CTEC93, cdiIEC93, cdiA-CTo1EC93 and cdiIo1EC93 transcripts (Figure 7). This analysis revealed that transcripts encoding orphan cdiA-CTo1EC93 and cdiIo1EC93 are expressed in wild-type EC93 cells, and that orphan message levels are very similar to those encoding the main cdiA-CTEC93 and cdiIEC93 (Figure 7). Addition- ally, the orphan transcript was expressed at approximately the same level in an EC93 strain deleted for the main cdiA-CTEC93/ cdiIEC93 region (Figure 7), indicating that the promoter driving orphan region transcription is upstream of the main cdiA-CTEC93. These results suggest that the orphan region is co-transcribed with the upstream cdiA and cdiI genes. Indeed, a large RT-PCR product was obtained with the forward cdiAEC93 and reverse cdiA-CTo1EC93 primers (data not shown), confirming that all of these ORFs are present on a single transcript. We next sought to detect the CdiA- CTo1EC93 protein by immunoblot using polyclonal antibodies raised against the homologous CdiA-CTUPEC536 from UPEC 536. Although this antiserum detects CdiA-CTo1EC93 produced from a heterologous expression system in E. coli K-12, we were unable to detect CdiA-CTo1EC93 in whole-cell lysates of EC93 (data not shown). In contrast to the orphan cdiA-CTo1EC93 fragment, the EC93 cdiIo1EC93 gene has an initiation codon and a properly spaced Shine-Dalgarno sequence, indicating that the CdiIo1EC93 protein is likely to be synthesized in EC93 cells. To determine if orphan CredsiisItoa1nEtC9t3o isCiDndIemededeixapteredssbedy, twhee tcehsitmederwicheCthdeiArEECC939-3CcTeoll1sECar9e3 protein. Because EC93 cells are CDI+, the chimeric inhibitor strain must itself be immune to EC93-mediated CDI. Therefore, we introduced a cosmid encoding the CdiAEC93-CTo1EC93 chimera into EC93 and used the resulting strain as the inhibitor for these experiments. Wild-type EC93 was not inhibited by EC93 expressing the CdiAEC93-CTo1EC93 chimera, but the EC93 DcdiA- CTo1EC93/DcdiIo1EC93 strain was inhibited approximately 103-fold (Figure 8). This growth inhibition was completely abrogated when orphan CdiIo1EC93 immunity protein was expressed from a plasmid in the EC93 DcdiA-CTo1EC93/DcdiIo1EC93 cells (Figure 8). Taken together, these results demonstrate that functional CdiIo1EC93 immunity protein is produced from the orphan locus in EC93. rhs Loci Encode Orphan Toxin/Immunity Modules In the course of our bioinformatic analyses, we found that many CdiA-CT sequences share significant identity with the C-terminal regions of Rhs proteins (Table S1). For example, the CdiA- CT91001(I) of Y. pestis Microtus strain 91001 (Q74T84) is similar to the C-terminal region of an Rhs/YD-repeat protein from Waddlia chondrophila WSU 86–1044 (D6YTT8) (Table S1 and Figure S7). The W. chondrophila rhs gene is followed by a small ORF that encodes a protein with similarity to CdiI91001(I) from Y. pestis Microtus 91001 (27% identity over 48 residues), suggesting this locus encodes a toxin/immunity protein pair. Intriguingly, Rhs- CT sequences are variable and demarcated by a well-conserved PLoS Genetics | www.plosgenetics.org 6 August 2011 | Volume 7 | Issue 8 | e1002217