Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI + bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.bacterial cell envelope | DNase activity | genetic selection | toxin/immunity genes | tRNase activity

For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/o-xylene monooxygenase hydroxylase is a dioxygen-activating enzyme. Structural analysis suggests two possible pathways for dioxygen access through the α-subunit to the diiron center: a channel or a series of hydrophobic cavities. To distinguish which is utilized as the O 2 migration pathway, the dimensions of the cavities and the channel were independently varied by site-directed mutagenesis and confirmed by X-ray crystallography. The rate constants for dioxygen access to the diiron center were derived from the formation rates of a peroxodiiron(III) intermediate, generated upon treatment of the diiron(II) enzyme with O 2 . This reaction depends on the concentration of dioxygen to the first order. Altering the dimensions of the cavities, but not the channel, changed the rate of dioxygen reactivity with the enzyme. These results strongly suggest that voids comprising the cavities in toluene/o-xylene monooxygenase hydroxylase are not artifacts of protein packing/folding, but rather programmed routes for dioxygen migration through the protein matrix. Because the cavities are not fully connected into the diiron active center in the enzyme resting state, conformational changes will be required to facilitate dioxygen access to the diiron center. We propose that such temporary opening and closing of the cavities may occur in all bacterial multicomponent monooxygenases to control O 2 consumption for efficient catalysis. Our findings suggest that other gas-utilizing enzymes may employ similar structural features to effect substrate passage through a protein matrix.A large number of metalloenzymes utilize dioxygen as a substrate. Understanding the process by which O 2 gains access to the active sites in these proteins has been a great challenge. Common substrates in biological systems, such as protons and electrons, require specific environments to facilitate translocation (1-4). By contrast, gaseous substrates like dioxygen may quickly diffuse through a protein matrix without direct assistance of specific local residues. Several enzymes that utilize gas molecules, such as O 2 (5, 6), H 2 (6, 7), or CO (8), as substrates have been investigated to understand how these small substances traverse the protein to reach their active sites. Most studies depended primarily on X-ray crystallography combined with Xe pressurization experiments to determine hydrophobic voids within the protein architecture that can be utilized to delineate substrate passage. The strong electron density and similar van der Waals diameter of xenon (4.3 Å) compared to that of O 2 (3.0-4.3 Å) renders it a useful surrogate for visualizing possible dioxygen routes within proteins by X-ray crystal structure analysis (5, 7). Because small gaseous molecules bind in a nonselective manner to the hydrophobic cavities in a protein, however, further evidence is required...

Highlights d The crystal structures of two CDI toxin,immunity protein complexes are presented d Both toxins are isoacceptor-specific ribonucleases that cleave deacylated tRNA GAU Ile d The immunity proteins do not share any sequence or structural homology d Similarities between these tRNase toxins likely arose through convergent evolution

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDIand CdiA-CT 1026b are both tRNases; however, each nuclease cleaves tRNA at a distinct position. We used a molecular docking approach to model each toxin bound to tRNA substrate. The resulting models fit into electron density envelopes generated by small-angle x-ray scattering analysis of catalytically inactive toxin domains bound stably to tRNA. CdiA-CT E479 is the third CDI toxin found to have structural homology to the PD(D/E)XK superfamily. We propose that CDI systems exploit the inherent sequence variability and active-site plasticity of PD(D/E)XK nucleases to generate toxin diversity. These findings raise the possibility that many other uncharacterized CDI toxins may belong to the PD(D/E)XK superfamily.

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.