A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

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Bacteria have developed mechanisms to communicate and compete with one another in diverse environments1. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli2. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional small immunity protein (CdiI) protects CDI+ cells from autoinhibition2,3. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.

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Figure 1: Analysis of CdiA chimaeras.
Figure 2: CdiA-CT contains growth inhibitory activity.
Figure 3: CdiI immunity protein binds specifically to cognate CdiA-CT and blocks activity.
Figure 4: CDI systems function in intrastrain growth competition.


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We thank A. Charkowski, A. Collmer, J. Roth and H. Schweizer for plasmids, bacterial strains and helpful discussions; W. Lathem for Y. pestis CO92 DNA; and R. Christoffersen for helpful advice on plant experiments. This work was supported by National Science Foundation grant 0642052 (D.A.L.), a Tri-Counties Blood Bank Postdoctoral Fellowship (S.K.A.) and National Institutes of Health grants GM078634 (C.S.H.), AI043986 (P.A.C.) and U54AI065359 (D.A.L., P.A.C. and C.S.H.). The content is the sole responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. This project made use of preliminary sequences from the Dickeya dadantii 3937 genome project supported by the Initiative for Future Agriculture and Food Systems grant no. 2001-52100-11316 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service.

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D.A.L., S.K.A., P.A.C. and C.S.H. designed the research. D.A.L., C.S.H., P.A.C., S.J.P. and S.K.A. prepared the manuscript. S.J.P. and B.R.B. performed bioinformatic analyses. B.R.B., A.M.J. and P.A.C. obtained initial evidence for the toxic nature of CdiA-CT, variability of CdiA-CTs and CdiIs, and binding between CdiA-CTs and cognate CdiIs in studies of Burkholderia pseudomallei cdi genes. S.K.A. cloned and performed competition assays and growth curves involving cdiBAI, cdiA-CT and cdiA chimaeras and deletions. B.A.B. and J.S.W. conducted the deletion mapping study, with assistance from S.K.A. B.A.B. and S.K.A. conducted the bacterial two-hybrid study. C.T.R. constructed D. dadantii cdi mutants and plasmids and performed growth competition assays on chicory. E.J.D. cloned, purified protein and performed the in vitro protein interaction and CdiA-CT activity studies.

Correspondence to David A. Low.

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Aoki, S., Diner, E., de Roodenbeke, C. et al. A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria. Nature 468, 439–442 (2010) doi:10.1038/nature09490

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