Author Summary Recent work from our laboratories has shown that many bacteria express contact-dependent growth inhibition (CDI) systems in which stick-like proteins on the cell surface deliver toxic tips into target cells. Over 60 distinct toxic tips have been identified in bacteria, and our data indicate that each CDI+ cell expresses a specific immunity protein that binds to its cognate toxin and inactivates it to prevent cell suicide. Here we identify genes for toxic tips that are not attached to the stick protein. Each of these ‘‘orphan’’ tips has toxic activity, which is blocked by its associated immunity protein. Remarkably, the orphan tips of some bacterial species are often found on the stick proteins in other species, suggesting that cells load and deliver different tips. We also report on a system called Rhs, which encodes another predicted stick-like protein that also carries variable tips. We found that the tips of Rhs proteins are toxic and that Rhs systems encode immunity proteins that specifically block toxin activity. CDI and Rhs toxin tip diversity may represent a microbial arms race, driven by the competition for environmental resources. distinct toxic nuclease activities, and that the corresponding CdiI proteins bind to their cognate CdiA-CT and block nuclease activity both in vitro and in vivo [5]. Thus, CdiA-CT/CdiI pairs constitute a polymorphic family of toxin/immunity modules that allow CDI systems to deploy a wide variety of growth inhibition activities. CdiA proteins share a number of characteristics with the Rhs protein family. The rhs genes were first identified in E. coli by C.W. Hill and colleagues, and were named rearrangement hotspots based on their role in chromosome duplications [10,11]. Rhs proteins are widely distributed throughout the eubacteria, but their function is poorly understood. Like CdiA, Rhs proteins are large, ranging from ,1,500 residues in Gram-negative bacteria to over 2,000 residues in some Gram-positive species. Rhs proteins also possess a central repeat region, though the characteristic YD peptide repeats of Rhs proteins are unrelated to the hemagglutinin repeats in CdiA. Moreover, Rhs proteins have variable C-terminal domains that are sharply demarcated by a conserved peptide motif (PxxxxDPxGL in the Enterobacteriaceae) [12]. Remarkably, we find that some CdiA and Rhs proteins share related C-terminal sequences, suggesting the protein families may be functionally analogous. Rhs proteins from a number of species appear to be exported to the cell surface [13–15], consistent with a role in cell- to-cell communication. Here we show that many CDI systems have an unusual genetic organization similar to that described for some Rhs loci [12]. Downstream of the cdiBAI genes, CDI systems often contain fragmentary gene pairs that resemble cdiA-CT/cdiI toxin/immu- nity modules. The predicted cdiA-CT fragments generally lack translation initiation signals but encode the VENN peptide motif that demarcates the CdiA-CT region in full-length CdiA proteins. These ‘‘orphan’’ CdiA-CT proteins possess growth inhibitory activities, which are specifically neutralized by the corresponding orphan CdiI immunity proteins. Moreover, we show that the orphan cdiA-CT/cdiI region is actively transcribed in E. coli EC93. Although the orphan CdiA-CT does not appear to be synthesized, functional orphan CdiI immunity protein is produced in EC93. We also show that the Rhs systems of D. dadantii 3937 encode toxin/immunity pairs. Rhs-CTs from D. dadantii 3937 inhibit cell growth when expressed in E. coli, and this toxic activity is specifically neutralized by the cognate RhsI protein encoded Toxin Effectors of CDI and Rhs Systems immediately downstream. These results suggest that Rhs consti- tutes another class of cell-surface proteins involved in intercellular competition, and that orphan CT/immunity modules may represent a reservoir of toxin/immunity diversity for both CDI and Rhs systems. Results Many CDI Systems Contain Orphan cdiA-CT/cdiI Gene Pairs Examination of the cdi locus in E. coli EC93 revealed two short open reading frames (ORFs) immediately downstream of the cdiI immunity gene (Figure 1). The first ORF lacks a translation initiation codon but encodes the VENN motif that typically demarcates variable CdiA-CT regions (Figure 1), suggesting the first ORF encodes a detached CdiA-CT remnant and the second ORF is its associated cdiI gene. A TBLASTN search of bacterial genomes revealed that the encoded proteins are related to the CdiA-CT/CdiI toxin/immunity pair from E. coli UPEC 536 (Figure S1). Thus, the cdiBAI gene cluster in E. coli EC93 is followed immediately by an ‘‘orphan’’ cdiA-CT/cdiI module related to the cdi locus of a different E. coli strain. To differentiate these modules from main cdiBAI clusters, we indicate orphan genes with a subscripted ‘‘o’’ and a number that indicates the position of the module in the cdi locus. Additionally, throughout the text we will indicate bacterial strains as superscripts. According to this nomenclature, the genes in the EC93 orphan module are designated cdiA-CTo1EC93 and cdiIo1EC93. Examination of CDI regions in other bacteria shows that orphan cdiA-CT/cdiI pairs are quite common. Although some CDI systems are comprised solely of the cdiBAI gene cluster, many loci are closely followed by one or more cdiA gene fragments that usually encode the VENN peptide motif (Figure 2). These orphan cdiA-CT genes are typically followed by small ORFs representing potential cdiI immunity genes. For example, the region II CDI system in Yersinia pseudotuberculosis PB1/+ contains four additional cdiA-CT gene fragments encoding the VENN motif, each associated with a putative cdiI gene (Figure 2 and Figure S2A). Three of these gene fragments (cdiA-CTo2PB1(II), cdiA-CTo3PB1(II) and cdiA-CTo4PB1(II)) share significant regions of homology with the upstream cdiAPB1(II) gene. The extent of these homologous regions varies between orphans, but the homology to full-length cdiAPB1(II) is limited to regions upstream of the VENN encoding sequences for cdiA-CTo2PB1(II) and cdiA-CTo3PPBB11(I(III))(FgeignuereshSa2reasnndoFsiiggunrieficSa3n)t. In contrast, the orphan cdiA-CTo1 identity with the full-length cdiAPB1(II) gene beyond the sequence encoding the VENN peptide (Figure S2C). Downstream of the VENN encoding region, the orphan cdiA-CToPB1(II) genes are unrelated to one another, but have homology to cdiA genes and cdiA-CT fragments from other bacteria. The predicted orphan CdiA-CTo1PB1(II) (UniProt B2K3A6) is related to CdiA-CTs from E. coli A0 34/86 (Q1RPM1; 87% identity over 107 residues) and Enterobacter cloacae subsp. cloacae ATCC 13047 (D5CBA0; 50% identity over 183 residues), as well as to orphan CdiA-CTs from Dickeya zeae Ech1591 (C6CGV6; 88% identity over 111 residues) and Citrobacter rodentium ICC168 (D2TJP2; 86% identity over 107 residues). Orphan CdiA-CTo2PB1(II) (B2K3A4) is related to the CdiA-CT from Serratia proteamaculans 568 (A8GK56; 63% identity over 131 residues). Orphan CdiA-CTo3PB1(II) (B2K3A2) is related to a CdiA-CT from Erwinia amylovora CFBP1430 (D4HWF3; 63% identity over 127 residues) and to orphan CdiA-CTs from E. coli EC869 (B3BM80; 75% identity over 297 residues) and Neisseria meningitidis MC58 (Q9K0T4; 57% identity over 136 residues). Finally, orphan CdiA-CTo4PB1(II) (B2K3A0) is related to the CdiA- PLoS Genetics | www.plosgenetics.org 2 August 2011 | Volume 7 | Issue 8 | e1002217