Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNAGAUIle. Although the toxins are similar in structure and substrate specificity, CdiA-CTKp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CTEC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiIKp342 forms a dimeric β sandwich, whereas CdiIEC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence.

Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiIYkris complex from Yersinia kristensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistent with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiIYkris binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase A-like toxins are commonly deployed in inter-bacterial competition.

Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact-dependent growth inhibition toxin CdiA-CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His-His-Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate-specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N-terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA-CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2 A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter-bacterial toxin delivery systems.