Bacteria have developed complex mechanisms to thrive in their environments. Contact- dependent growth inhibition (CDI), a novel inter-bacterial competition mechanism was recently discovered in gram-negative bacteria, and is used to block growth of neighboring cells. The CDI growth inhibitory activity is contained in the C-terminal domain of CdiA effector proteins (CdiA- CT, toxin), and is delivered into target bacterial cells. All CDI+ bacteria also produce small CdiI immunity proteins that protect them from autoinhibition. CdiA-CT and CdiI from different species exhibit high sequence variability, which predicts diverse structures and/or interaction interfaces. Here we describe the initial structural characterization of the CDI system, providing functional insights into distinct CDI mechanisms.

We have solved X-ray crystal structures of CDI toxin/immunity complexes from Escherichia coli (EC869), Burkholderia pseudomallei (1026b), and Enterobacter cloacae (ECL), representing three distinct families of CDI proteins. The interfaces between the toxin/immunity pairs are distinct, providing important insights into CdiA-CT/CdiI binding specificity. The EC869 CdiA-CT/CdiI binding interface is especially intriguing and consists of a unique β-augmentation interaction, by which CdiA-CT^EC869 inserts a short β-hairpin structural element into a cavity on the surface of CdiI^EC869. This interface is conserved among EC869 family CdiA-CT/CdiI complexes, but heterologous toxin and immunity proteins within this family do not interact with high affinity. Additionally, the EC869, 1026b, and ECL toxin structures helped elucidate and characterize three distinct CdiA-CT growth-inhibiting activities. EC869 and 1026b CdiA-CTs are structurally similar and contain DNase and tRNase activities, respectively. In contrast, ECL CdiA-CT shares no structural homology with EC869 or 1026b CdiA-CTs, but is structurally and functionally similar to colicin E3, a toxin with rRNase activity from a distinct bacterial system. We also report the crystal structure of the CdiA-CT toxin from uropathogenic E. coli strain 536 in complex with its permissive factor, CysK, representing a unique step in CDI. The complex formed mimics the interaction found in the cysteine synthase complex. Finally, we solved the structure of a domain from the large, non-toxin region of E. coli CdiA, providing a starting point towards future research into the CdiA-CT cleavage and translocation mechanisms.