Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems Stephen J. Poole1*, Elie J. Diner2, Stephanie K. Aoki1, Bruce A. Braaten 1 , Claire t’Kint de Roodenbeke1, David A. Low1,2, Christopher S. Hayes1,2 1 Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America, 2 Biomolecular Science and Engineering Program, University of California Santa Barbara, Santa Barbara, California, United States of America Abstract Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA- CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These ‘‘orphan’’ cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems. Citation: Poole SJ, Diner EJ, Aoki SK, Braaten BA, t’Kint de Roodenbeke C, et al. (2011) Identification of Functional Toxin/Immunity Genes Linked to Contact- Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems. PLoS Genet 7(8): e1002217. doi:10.1371/journal.pgen.1002217 Editor: Mark Achtman, Environmental Research Institute, University College Cork, Ireland Received January 31, 2011; Accepted June 17, 2011; Published August 4, 2011 Copyright: 2011 Poole et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health through grant U54 AI065359 (DAL and CSH), National Science Foundation grant 0642052 (DAL), and Tri-Counties Blood Bank Postdoctoral Fellowship (SKA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: poole@lifesci.ucsb.edu Introduction Many bacteria lead social lives in communities where they cooperate and compete with members of their own species, as well as those of other species [1]. One mechanism of bacterial communication is quorum sensing, in which small signaling molecules are released to coordinate group behavior when a critical cell density has been attained [2]. Other modes of communication based on direct cell-to-cell contact have recently been identified in bacteria. Contact-dependent signaling helps to coordinate cell aggregation and fruiting body formation in Myxococcus xanthus [3], and is also exploited to inhibit the growth of neighboring cells. Contact-dependent growth inhibition (CDI) was first described in the Escherichia coli isolate EC93 [4], and has subsequently been demonstrated in Dickeya dadantii 3937 [5]. CDI is mediated by the CdiB-CdiA two-partner secretion system. CdiB is a predicted outer membrane b-barrel protein that is required for secretion and presentation of the CdiA exoprotein on the cell surface [6,7]. Like other two-partner secretion exoproteins, CdiA contains an N-terminal transport domain followed by a hemag- glutinin repeat region that is predicted to adopt an extended filamentous b-helical structure [7–9]. The CDI growth inhibitory activity resides within the C-terminus of CdiA (CdiA-CT). The cdi locus also encodes a small CdiI immunity protein immediately downstream of cdiA. CdiI protects EC93 cells from CdiA-mediated growth autoinhibition [5]. CDI systems are widespread amongst a-, b-, and c-proteobac- teria [5]. CdiA exoproteins are related throughout most of their length, which varies from 1,400 to 2,000 amino acid residues in Neisseria and Moraxella species to over 5,600 residues for some Dickeya and Pseudomonas strains [5]. However, the CdiA-CT regions are highly variable, with CdiA sequences diverging abruptly after a VENN peptide motif found within the conserved DUF638 domain (Pfam PF04829). Similarly, CdiI sequences are also highly variable, suggesting that these immunity proteins specifically bind to cognate CdiA-CTs and neutralize their toxic activities. In support of this model, we recently showed that CdiA-CTs from Dickeya dadantii 3937 and uropathogenic E. coli (UPEC) 536 possess PLoS Genetics | www.plosgenetics.org 1 August 2011 | Volume 7 | Issue 8 | e1002217