Toxin Effectors of CDI and Rhs Systems Figure 9. Rhs genes have variable CT encoding sequences, immunity genes, and orphan rhs-CT/rhsI modules. ACT comparison of related Rhs regions from Y. pseudotuberculosis strains IP31758 (top) and IP32953 (bottom). This region is highly conserved between the two strains, but the rhs genes encode unrelated CT sequences and the adjacent rhsI genes are unrelated. Both strains contain a related orphan rhs-CT/rhsI module. The orphan module in strain IP32953 has more upstream rhs coding sequence, but contains an in-frame stop codon in this retained sequence. The vgrG gene is a conserved component of Type VI secretion systems; TPR is a conserved gene encoding a potential tetratricopeptide repeat protein; and X and Y are conserved predicted genes of unknown function. doi:10.1371/journal.pgen.1002217.g009 followed by an array of orphan cdiA-CT/cdiI modules. These arrays can be extensive, with eleven orphan gene pairs in the cdi locus of E. coli EC869. The widespread occurrence of orphan cdiA- CT/cdiI modules in diverse bacterial species argues that these gene pairs confer a selective advantage. Although the majority of orphan cdiA-CT genes lack translation initiation signals, orphan cdiI genes appear to be fully capable of expression. Therefore, bacteria could maintain orphan modules to build a repertoire of immunity genes to protect themselves from neighboring bacteria that express diverse CdiA proteins. Broad range immunity would clearly confer an advantage and our data demonstrate that EC93 does in fact express its orphan CdiI immunity protein. However, if bacteria are collecting immunity genes, then it is unclear why the toxin-encoding cdiA-CT fragments are retained in the process. One model to explain these findings is that orphan modules allow the bacterium to change its toxin/immunity profile by recombination between the highly conserved VENN-encoding sequences. This may have occurred in the cdi locus of Y. pestis Microtus 91001, where a large deletion has loaded an orphan cdiA-CT/cdiI module onto the main cdiA gene. However, such a strategy for generating CdiA-CT diversity could have serious shortcomings. First, although reloading CdiA with a new toxic C-terminal domain would produce a novel weapon, the recombination would also delete cdiI and render the cell susceptible to the original CdiA protein expressed by neighboring bacteria that have not recombined their cdi locus. Second, simple recombination between cdiA and distal orphan cdiA-CT genes would delete all intervening cdiA-CT/cdiI modules. An alternative model is that recombination between cdiA (or rhs) and the orphan regions occurs following tandem duplication of the loci. This mechanism would allow cdiA- CT/cdiI modules to be rearranged without the loss of immunity or genetic diversity, because a copy of the original cdi/rhs system would be present [22,23]. The results presented here have also revealed a connection between CDI and Rhs systems. The rhs genes were first described in E. coli, and were originally thought to be rearrangement hot spots because of a recombination event between nearly identical sequences within the rhsA and rhsB genes [10,11]. These proteins are widely distributed throughout the eubacteria and related proteins containing YD peptide repeats are also found in metazoans including all vertebrates. Despite their prevalence, the function of these proteins is almost completely unknown. Our data show that at least some Rhs systems encode toxin/immunity protein pairs, and a recent bioinformatic study has proposed that Rhs proteins contain toxic nuclease domains [24]. These results are consistent with work from Hill and colleagues showing that the C-terminal region of E. coli RhsA blocks the recovery of stationary phase cells [20]. Moreover, this growth inhibition was neutralized by expression of the ORF (yibA) encoded immediately downstream of rhsA [20]. More recently an Rhs-related protein from Pseudomonas savastanoi pv. savastanoi was found to be associated with bacteriocin activity [25]. Because of the parallels between CDI and Rhs, we suspect that Rhs proteins are also exported to block the growth of neighboring cells and thereby impart a competitive advantage to the inhibitor cell. However, many Rhs proteins from Gram-negative bacteria lack recognizable signal sequences, so the export pathway is unclear in several instances. Many rhs genes are linked to valine-glycine repeat (Vgr)-encoding genes that are associated with Type VI secretion systems [26]. VgrG proteins associated with Type VI secretion systems have structural similarity to bacteriophage cell-puncturing proteins [27,28], and are therefore ideal for penetrating bacterial envelopes. Indeed, recent work has demonstrated that Type VI systems are used to deliver protein toxins into target bacteria [29]. Whether the close genetic linkage between rhs and vgr genes extends to a functional relationship remains to be determined. Although the Rhs systems examined here appear to be growth inhibitory, there are indications that Rhs proteins have other signaling modalities. Youdarian and Hartzell found that an Rhs- related YD repeat protein (Q1CXS7) from Myxococcus xanthus plays an important role in social motility [15], which occurs when individual bacteria make contact with one another to coordinate cell movement. The M. xanthus YD repeat protein has a signal sequence and is presumably exported to the surface. The gene encoding Q1CXS7 (MXAN_6679) is closely followed by a small ORF that is suggestive of an rhsI gene. However, it is not clear that immunity would be required in this signaling pathway. Perhaps this small ORF encodes a peptide that is involved in receiving the PLoS Genetics | www.plosgenetics.org 9 August 2011 | Volume 7 | Issue 8 | e1002217