variicola At-22 and Erwinia pyrifoliae DSM 12163 both contain cdiA- CT fragments that do not encode VENN and lack associated cdiI genes (Figure 2). In several instances, the orphan cdiA-CT genes are disrupted by IS elements and transposon genes. For example, the orphan cdiA-CTo9EC869 from E. coli EC869 is interrupted by an SSuT antimicrobial resistance element [16]. Orphan cdiA-CT fragments can retain varying amounts of cdiA sequence upstream of the VENN-encoding region, but in some cases these homologous sequences are absent (Figure 2). For example, the orphan cdiA-CTo7EC869 gene from E. coli EC869 is unrelated to the adjacent cdiA-CToEC869 fragments, but is almost identical (99% identity from the VENN region onward) to the orphan cdiA- CTo1254 from E. coli strain 254 (Figure S4). Moreover, the associated cdiI immunity genes are also nearly identical. A comparison of the E. coli EC869 and E. coli strain 254 genomes shows that the homology between these orphan cdiA-CT/cdiI modules begins at the VENN encoding sequence and extends precisely to the VENN sequence of the next cdiA-CT orphan (Figure S4). Interchange of cdiA-CT/cdiI Modules All sequenced Y. pestis strains share two large blocks of conserved DNA that contain cdi loci. For the region I CDI system (positioned between the mannitol transporter and dioxygenase b- subunit genes), the predicted CdiA(I) protein is identical in all fully sequenced Y. pestis strains with the exception of the Microtus 91001 strain. CdiA(I) sequences N-terminal to the VENN motif are essentially identical in Microtus 91001 and other Y. pestis strains, though there is a six amino acid residue deletion within the hemagglutinin repeat region of Microtus 91001. However, following the VENN motif, the CdiA-CT91001(I) of Microtus 91001 diverges from that of other Y. pestis strains (Figure S5). Pairwise comparison of the Y. pestis CO92 and Y. pestis Microtus 91001 genomes revealed a 3,557 base-pair deletion in the Microtus 91001 region I cdi locus that has fused an orphan cdiA- CT/cdiI module to the upstream cdiA91001(I) gene (Figure 3A), producing a CdiA/CdiI toxin/immunity pair that is different from other Y. pestis strains. Thus, the CdiA91001(I) protein from Microtus 91001 contains an orphan CdiA-CT effector domain from the CO92 strain. Another possible example of CdiA-CT interchange is found in the region II CDI system (located between tellurium resistance genes and a predicted autotransporter) shared by Y. pseudotubercu- losis PB1/+ and IP31758 strains. The main cdiA-CTIP31758(II) and cdiIIP31758(II) sequences of Y. pseudotuberculosis IP31758 are essen- tially identical to the cdiA-CTo4PB1(II)/cdiIo4PB1(II) orphan module from strain PB1/+ (99% identity over 3,646 nucleotides) (Figure 3B). Additionally, comparison of these loci revealed other complex rearrangements. The orphan cdiA-CTo1PB1(II)/cdiIo1PB1(II) module of strain PB1/+ is nearly identical to cdiA-CTo2IP31758(II)/ cdiIo2IP31758(II) from IP31758 (98% identity over 2,656 nucleotides), and the orphan cdiA-CTo2PB1(II)/cdiIo2PB1(II) from PB1/+ is 98% identical (over 1,073 nucleotides) to a cdiA gene fragment (which lacks the VENN encoding sequence) and its associated cdiI gene in strain IP31758 (Figure 3B). There are at least two possible explanations for these observations. The two Y. pseudotuberculosis strains could have independently acquired the same modules and incorporated them at different sites within the cdi locus. Alternatively, the cdi locus of a common ancestor may have rearranged during strain diversification. Although the mechanisms underlying these complex exchanges are unknown, these obser- vations suggest that orphan cdiA-CT fragments are a potential source of CdiA/CdiI toxin/immunity diversity. Toxin Effectors of CDI and Rhs Systems Orphan cdiA-CT/cdiI Modules Are Functional If orphan cdiA-CT/cdiI modules are merely unused, nonfunc- tional remnants of full-length cdiA/cdiI genes, then there should be no selective pressure to maintain CdiA-CT toxin activity and CdiI immunity function. To determine whether orphan cdiA-CT/cdiI modules encode functional proteins, we characterized the orphan CdiA-CT/CdiI proteins from E. coli EC93. As described above, CdiA-CTo1EC93 and CdiIo1EC93 are very similar to the UPEC 536 CdiA-CTUPEC536/CdiIUPEC536 pair that we have previously characterized [5]. We first tested whether CdiA-CTo1EC93 and CdiIo1EC93 bind to one another as predicted for a toxin/immunity pair. We introduced a translation initiation signal upstream of the cdiA-CTo1EC93 fragment and co-expressed orphan CdiA-CTo1EC93 with CdiIo1EC93 immunity protein carrying a hexa-histidine (His6) epitope tag at its C-terminus. Ni2+-affinity purification of His6- tagged CdiIo1EC93 under non-denaturing conditions resulted in co- purification of CdiA-CTo1EC93 (data not shown), demonstrating that the two proteins bind each other. Given the similarity between CdiA-CTo1EC93 and CdiA-CTUPEC536, we next tested whether the CdiIUPEC536 and CdiIo1EC93 immunity proteins are able to bind near-cognate CdiA-CTs. His6-tagged CdiI proteins were first separated from their cognate CdiA-CT proteins by Ni2+- affinity chromatography under denaturing conditions. The individual proteins were then refolded and tested for binding to their cognate partners. Refolded CdiA-CTo1EC93 and CdiA- CTUPEC536 were able re-bind to their cognate CdiI proteins (Figure 4A). However, the His6-tagged CdiI proteins were unable to bind stably to the near-cognate CdiA-CTs (Figure 4A). These results show that the EC93 orphan CdiA-CT/CdiI proteins physically interact with one another, but appear to have diverged enough from the UPEC 536 system that high-affinity binding between the two systems no longer occurs. We previously demonstrated that CdiA-CTUPEC536 is a nuclease that cleaves tRNA [5]; therefore we asked whether CdiA- CTo1EC93 also possesses this biochemical activity. Purified CdiA- CTo1EC93 cleaved a number of different tRNA species in a manner that was indistinguishable from CdiA-CTUPEC536 tRNase activity (Figure 4B and data not shown). This tRNase activity was inhibited by the addition of equimolar His6-tagged CdiIo1EC93 (Figure 4B). Intriguingly, His6-tagged CdiIUPEC536 was also able to neutralize the tRNase activity of CdiA-CTo1EC93, but CdiIo1EC93 had no effect on CdiA-CTUPEC536 activity (Figure 4B). Presum- ably, CdiIUPEC536 interacts with CdiA-CTo1EC93, but this binding is not of sufficient affinity to allow co-purification by Ni2+-affinity chromatography. Together, these results show that the EC93 orphan CdiA-CT/CdiI proteins retain the biochemical features of a functional toxin/immunity module. Orphan CdiA-CTs Inhibit Cell Growth We next asked whether the EC93 orphan CdiA-CT retains growth inhibitory activity. In general, cdiA-CT gene fragments are very toxic and can only be maintained on plasmids if the cognate cdiI immunity gene is also present. However, CdiI immunity proteins efficiently block CdiA-CT activity, making it difficult to assess CdiA-CT toxicity. To circumvent this problem, we used the controllable proteolysis system of McGinness and Sauer [17] to activate CdiA-CTo1EC93 through degradation of the CdiIo1EC93 immunity protein. This strategy uses the SspB adaptor protein to deliver ssrA(DAS) peptide-tagged proteins to the ClpXP protease. We tagged the C-terminus of CdiIo1EC93 with the ssrA(DAS) peptide and co-expressed it with CdiA-CTo1EC93 in E. coli DsspB cells. Degradation of tagged CdiIo1EC93 was then initiated by induction of SspB synthesis from a plasmid-borne arabinose- inducible promoter, resulting in growth arrest after approximately PLoS Genetics | www.plosgenetics.org 4 August 2011 | Volume 7 | Issue 8 | e1002217