Figure 10. The Rhs genes of D. dadantii 3937 encode toxin/ immunity pairs. E. coli sspB+ cells expressing RhsI3937 proteins were incubated with supercoiled plasmids encoding the various Rhs-CT3937/ RhsI3937-DAS pairs and plated to select stable transformants. All Rhs- CT3937/RhsI3937-DAS constructs were also introduced into E. coli DsspB cells to demonstrate that RhsI3937-DAS degradation is required for growth inhibition. doi:10.1371/journal.pgen.1002217.g010 signal from the C-terminal region of the YD repeat protein. Recent work on the entomopathogen Pantoea stewartii suggests another function for a class of Rhs-related proteins termed Ucp (for you cannot pass) [30]. The P. stewartii genome contains seven ucp homologs that encode large proteins (1,200–1,300 residues) with similar N-terminal regions and variable C-terminal sequenc- es. The Ucp1 protein mediates bacterial aggregation and is a virulence factor required for pathogenicity against the aphid host. It was postulated that Ucp1 and other Ucp proteins function primarily as adhesins and that C-terminal variability is driven by the need to evade host immune responses [30]. Our results suggest an alternative possibility. The C-terminal peptide of Ucp1 could be delivered into insect cells and exert a toxic effect in a manner Toxin Effectors of CDI and Rhs Systems similar to that proposed for CDI [5]. In this model, the other six Ucp proteins could be targeted against other bacterial competitors or different eukaryotic hosts. Finally, another intriguing example of Rhs signaling has been proposed for teneurins. The teneurin protein family is comprised of four paralogs that are present in all vertebrates [31]. These proteins are type II integral membrane proteins (single transmem- brane span with the C-terminus presented extracellularly) that play a role in axon guidance and neural patterning during development [31,32]. Like Rhs proteins, the teneurins are large (2,500–2,800 residues) and the C-terminal half is comprised of several YD peptide repeats. Remarkably, all teneurins contain a C-terminal associated peptide (TCAP) that is similar to neuroen- docrine signaling peptides [33,34]. The TCAP region is adjacent to a phylogenetically conserved furin protease cleavage site (RxRR) [31], suggesting that the TCAPs are released and enter target cells to exert neuromodulatory effects. Because some CdiA- CTs and Rhs-CTs have toxic nuclease activity, presumably they must also be cleaved for delivery into the cytoplasm of target cells. The parallel between CDI/Rhs-mediated growth inhibition and the proposed teneurin signaling pathway is striking, suggesting that intercellular communication through the delivery of cleaved C- terminal peptides is ubiquitous and possibly ancient. Perhaps these systems have arisen from a common YD-repeat protein ancestor. Materials and Methods Bioinformatic Analysis Pairwise sequence comparisons were performed using GCG Gap or pairwise BLAST. Related CdiA-CT, Rhs-CT, CdiI, RhsI and orphan sequences were found using TBLASTN against assembled bacterial genomes, because these regions are often not annotated and thus not represented in the non-redundant protein database. Pairwise comparisons of genomic regions were per- formed using WebACT (www.webact.org) with BLASTN com- parisons. The UniProt, GenBank and gene locus accession numbers for each full-length CdiA and Rhs protein discussed in this work are presented in Table S2. Plasmid Constructs The orphan cdiA-CTo1EC93/cdiIo1EC93 module from E. coli EC93 was amplified using oligonucleotides, EC93orph-Nco (59 - TTG CCA TGG AGA ATA ACT CGC TGA GC) and EC93orph- Spe (59 - ATC ACT AGT GGC ATT AGA TAG CTT ATC TAT TTT TGC) (restriction endonuclease sites are underlined), followed by digesting with NcoI and SpeI, and ligation to plasmid pET21S [5]. The resulting construct overproduces CdiA- CTo1EC93 and C-terminally His6-tagged CdiIo1EC93. The EC93 orphan immunity gene was amplified using primers #1658 (59 - CAA CAA GCA TGC CCC GAC TTT GAG ACC AGA ATA TC) and #1664 (59 - ATC AGG AGC ATG GTA TAT GAC AAC ATT TAG ATC). The resulting PCR product was digested with SphI and ligated to EcoRV/SphI digested plasmid pBR322 under control of the tet promoter. Orphan cdiA-CT/cdiI modules from D. dadantii 3937 and E. coli EC869 were amplified using 3937orph-Nco (59 - AAG CCA TGG TGG AGA ATA ACT ATC TGA GCA G) and 3937orph-Spe (59 - TCT ACT AGT AGG CTG GTA ATC TTC ATA TTC C); and EC869orph11-Nco (59 - ATT CCA TGG GCA CAA ACC AGT CTC TGA CCT TCG) and EC869orph11-Spe (59 - TCT ACT AGT ACC TTT GCA GCG ACT CAA GGC CAG), respectively. The rhs-CT3937/ rhsI3937 modules from D. dadantii 3937 were amplified using the following primer pairs: rhsB-Nco (59 - CAG CCA TGG AAA PLoS Genetics | www.plosgenetics.org 10 August 2011 | Volume 7 | Issue 8 | e1002217