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Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ∆VENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation., (© 2022. The Author(s).)

Contact-dependent growth inhibition (CDI) systems enable the direct transfer of protein toxins between competing Gram-negative bacteria. CDI + strains produce cell surface CdiA effector proteins that bind specific receptors on neighboring bacteria to initiate toxin delivery. Three classes of CdiA effectors that recognize different outer membrane protein receptors have been characterized in Escherichia coli to date. Here, we describe a fourth effector class that uses the lipopolysaccharide (LPS) core as a receptor to identify target bacteria. Selection for CDI-resistant target cells yielded waaF and waaP "deep-rough" mutants, which are unable to synthesize the full LPS core. The CDI resistance phenotypes of other waa mutants suggest that phosphorylated inner-core heptose residues form a critical CdiA recognition epitope. Class IV cdi loci also encode putative lysyl acyltransferases (CdiC) that are homologous to enzymes that lipidate repeats-in-toxin (RTX) cytolysins. We found that catalytically active CdiC is required for full target cell killing activity, and we provide evidence that the acyltransferase appends 3-hydroxydecanoate to a specific Lys residue within the CdiA receptor-binding domain. We propose that the lipid moiety inserts into the hydrophobic leaflet of lipid A to anchor CdiA interactions with the core oligosaccharide. Thus, LPS-binding CDI systems appear to have co-opted an RTX toxin-activating acyltransferase to increase the affinity of CdiA effectors for the target cell outer membrane. IMPORTANCE Contact-dependent growth inhibition (CDI) is a common form of interbacterial competition in which cells use CdiA effectors to deliver toxic proteins into their neighbors. CdiA recognizes target bacteria through specific receptor molecules on the cell surface. Here, we describe a new family of CdiA proteins that use lipopolysaccharide as a receptor to identify target bacteria. Target cell recognition is significantly enhanced by a unique fatty acid that is appended to the receptor-binding region of CdiA. We propose that the linked fatty acid inserts into the target cell outer membrane to stabilize the interaction. The CdiA receptor-binding region appears to mimic the biophysical properties of polymyxins, which are potent antibiotics used to disrupt the outer membranes of Gram-negative bacteria.

The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI ( cdiBAI ) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAI EC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system ( cdiBAIEC93-2 ) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI + bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.

The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secY S281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CT o10 EC869 or by CdiA-CT GN05224 , the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CT GN05224 was reduced in secY S281F target cells and absent in secY S281F Δ ppiD or secY S281F Δ yfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY ( secY G313W ) renders Δ ppiD or Δ yfgM target cells specifically resistant to CdiA-CT GN05224 but not to CdiA-CT o10 EC869 , further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins. IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes., (Copyright © 2021 Jones et al.)

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiA EC93 from Escherichia coli EC93 and OmpC-specific CdiA EC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiA EC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiA EC93 that carries the corresponding sequence from CdiA EC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads. IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads., (Copyright © 2017 Ruhe et al.)

Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT EC869 tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT EC869 binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT EC869 only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.

Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI., Competing Interests: The authors have declared that no competing interests exist

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability., Competing Interests: The authors declare no conflict of interest.

Contact-dependent growth inhibition (CDI) systems are widespread amongst Gram-negative bacteria where they play important roles in inter-cellular competition and biofilm formation. CDI+ bacteria use cell-surface CdiA proteins to bind neighboring bacteria and deliver C-terminal toxin domains. CDI+ cells also express CdiI immunity proteins that specifically neutralize toxins delivered from adjacent siblings. Genomic analyses indicate that cdi loci are commonly found on plasmids and genomic islands, suggesting that these Type 5 secretion systems are spread through horizontal gene transfer. Here, we examine whether CDI toxin and immunity activities serve to stabilize mobile genetic elements using a minimal F plasmid that fails to partition properly during cell division. This F plasmid is lost from Escherichia coli populations within 50 cell generations, but is maintained in ~60% of the cells after 100 generations when the plasmid carries the cdi gene cluster from E. coli strain EC93. By contrast, the ccdAB "plasmid addiction" module normally found on F exerts only a modest stabilizing effect. cdi-dependent plasmid stabilization requires the BamA receptor for CdiA, suggesting that plasmid-free daughter cells are inhibited by siblings that retain the CDI+ plasmid. In support of this model, the CDI+ F plasmid is lost rapidly from cells that carry an additional cdiI immunity gene on a separate plasmid. These results indicate that plasmid stabilization occurs through elimination of non-immune cells arising in the population via plasmid loss. Thus, genetic stabilization reflects a strong selection for immunity to CDI. After long-term passage for more than 300 generations, CDI+ plasmids acquire mutations that increase copy number and result in 100% carriage in the population. Together, these results show that CDI stabilizes genetic elements through a toxin-mediated surveillance mechanism in which cells that lose the CDI system are detected and eliminated by their siblings.

CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer., (© 2015 John Wiley & Sons Ltd.)

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.

Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI(+) inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm., (© 2014 John Wiley & Sons Ltd.)

Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition that requires direct cell-to-cell contact. CDI(+) inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C-terminal region (CdiA-CT). Here, we show that purified CdiA-CT(536) toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by-passing the requirement for cell-to-cell contact during toxin delivery. Genetic analyses demonstrate that the N-terminal domain of CdiA-CT(536) is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA-CT(536) import requires conjugative F pili. We provide evidence that the N-terminal domain of CdiA-CT(536) interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F(+) cells. We propose that CdiA-CT(536) mimics the pilin-binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction., (© 2014 John Wiley & Sons Ltd.)

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. CDI(+) cells bind to susceptible target bacteria and deliver a toxic effector domain derived from the carboxyl terminus of CdiA (CdiA-CT). More than 60 distinct CdiA-CT sequence types have been identified, and all CDI toxins characterized thus far display RNase, DNase, or pore-forming activities. CDI systems also encode CdiI immunity proteins, which specifically bind and inactivate cognate CdiA-CT toxins to prevent autoinhibition. CDI activity appears to be limited to target cells of the same species, suggesting that these systems play a role in competition between closely related bacteria. Recent work on the CDI system from uropathogenic Escherichia coli (UPEC 536) has revealed that its CdiA-CT toxin binds tightly to a cysteine biosynthetic enzyme (CysK) in the cytoplasm of target cells. The unanticipated complexity in the UPEC CDI pathway raises the possibility that these systems perform other functions in addition to growth inhibition. Finally, we propose that the phenomenon of CDI is more widespread than previously appreciated. Rhs (rearrangement hotspot) systems encode toxin-immunity pairs, some of which share significant sequence identity with CdiA-CT/CdiI proteins. A number of recent observations suggest that Rhs proteins mediate a distinct form of CDI.

Unlabelled: Bacteria that express contact-dependent growth inhibition (CDI) systems outcompete siblings that lack immunity, suggesting that CDI mediates intercellular competition. To further explore the role of CDI in competition, we determined the target cell range of the CDIEC93 system from Escherichia coli EC93. The CdiAEC93 effector protein recognizes the widely conserved BamA protein as a receptor, yet E. coli EC93 does not inhibit other enterobacterial species. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Consistent with this hypothesis, other enterobacteria are sensitized to CDIEC93 upon the expression of E. coli bamA and E. coli cells become CDIEC93 resistant when bamA is replaced with alleles from other species. Our data indicate that BamA loops 6 and 7 form the CdiAEC93-binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAEcoli and CdiAEC93 play a role in self-nonself discrimination., Importance: Contact-dependent growth inhibition (CDI) systems are widespread among Gram-negative bacteria, enabling them to bind to neighboring bacterial cells and deliver protein toxins that inhibit cell growth. In this study, we tested the role of CDI in interspecies competition using intestinal isolate Escherichia coli EC93 as an inhibitor cell model. Although E. coli EC93 inhibits different E. coli strains, other bacterial species from the intestine are completely resistant to CDI. We show that resistance is due to small variations in the CDI receptor that prevent other species from being recognized as target cells. CDI receptor interactions thus provide a mechanism by which bacteria can distinguish siblings and other close relatives (self) from more distant relatives or other species of bacteria (nonself). Our results provide a possible means by which antimicrobials could be directed to one or only a few related bacterial pathogens by using a specific receptor "zip code."

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

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., Competing Interests: The authors have declared that no competing interests exist.

Contact-dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer membrane beta-barrel proteins into the outer membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI+ inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, alpha-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cells binding and CDI. A second class of CDI-resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner membrane component of a multidrug efflux pump that normally forms a cell envelope-spanning complex with the membrane fusion protein AcrA and the outer membrane protein TolC. Strikingly, the requirement for the BamA and AcrB proteins in CDI is independent of their multi-component machines, and thus their role in the CDI pathway may reflect novel, import-related functions.

Bacteria have developed mechanisms to communicate and compete with each other for limited environmental resources. We found that certain Escherichia coli, including uropathogenic strains, contained a bacterial growth-inhibition system that uses direct cell-to-cell contact. Inhibition was conditional, dependent upon the growth state of the inhibitory cell and the pili expression state of the target cell. Both a large cell-surface protein designated Contact-dependent inhibitor A (CdiA) and two-partner secretion family member CdiB were required for growth inhibition. The CdiAB system may function to regulate the growth of specific cells within a differentiated bacterial population.

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.

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.

Bacteria have developed several strategies to communicate and compete with one another in complex environments. One important mechanism of inter-bacterial competition is contact-dependent growth inhibition (CDI), in which Gram-negative bacteria use CdiB/CdiA two-partner secretion proteins to suppress the growth of neighboring target cells. CdiB is an Omp85 outer-membrane protein that exports and assembles CdiA exoproteins onto the inhibitor cell surface. CdiA binds to receptors on susceptible bacteria and subsequently delivers its C-terminal toxin domain (CdiA-CT) into the target cell. CDI systems also encode CdiI immunity proteins, which specifically bind to the CdiA-CT and neutralize its toxin activity, thereby protecting CDI(+) cells from auto-inhibition. Remarkably, CdiA-CT sequences are highly variable between bacteria, as are the corresponding CdiI immunity proteins. Variations in CDI toxin/immunity proteins suggest that these systems function in bacterial self/non-self recognition and thereby play an important role in microbial communities. In this review, we discuss recent advances in the biochemistry, structural biology and physiology of CDI.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.