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Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs⁻ mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro–Ala–Ala–Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine–glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ΔrhsA ΔrhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ΔrhsA ΔrhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs⁺ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Rearrangement hotspot (Rhs) and related YD-peptide repeat proteins are widely distributed in bacteria and eukaryotes, but their functions are poorly understood. Here, we show that Gram-negative Rhs proteins and the distantly related wall-associated protein A (WapA) from Gram-positive bacteria mediate intercellular competition. Rhs and WapA carry polymorphic C-terminal toxin domains (Rhs-CT/WapA-CT), which are deployed to inhibit the growth of neighboring cells. These systems also encode sequence-diverse immunity proteins (RhsI/WapI) that specifically neutralize cognate toxins to protect rhs ⁺/wapA ⁺ cells from autoinhibition. RhsA and RhsB from Dickeya dadantii 3937 carry nuclease domains that degrade target cell DNA. D. dadantii 3937 rhs genes do not encode secretion signal sequences but are linked to hemolysin-coregulated protein and valine-glycine repeat protein G genes from type VI secretion systems. Valine-glycine repeat protein G is required for inhibitor cell function, suggesting that Rhs may be exported from D. dadantii 3937 through a type VI secretion mechanism. In contrast, WapA proteins from Bacillus subtilis strains appear to be exported through the general secretory pathway and deliver a variety of tRNase toxins into neighboring target cells. These findings demonstrate that YD-repeat proteins from phylogenetically diverse bacteria share a common function in contact-dependent growth inhibition.

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 identifiedptsG, metI, rbsC, gltK/gltJ, yciB,andftsHmutations 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.

This study significantly contributes to our knowledge of kin selection and social behavior in bacteria. The interactions between four homologous toxin–immunity protein systems of Myxococcus xanthus were investigated, and evidence was obtained that these systems can distinguish between self and nonself cells within a species. Importantly, this study revealed that nonself lineages, which display varying degrees of genetic relatedness, can co-grow and collaborate in distinct patterns. This discovery implies that the differential crosstalk between homologous toxin–immunity proteins can mimic the degree of kinship; through this activity, bacteria can differentiate close and distant relatives. This novel insight into bacterial social dynamics and kin discrimination supports kin selection theory and enriches our knowledge on microbial interactions and evolutionary strategies. These findings have broad implications for microbial ecology, evolution, and the development of cooperation strategies.

The type VI secretion system (T6SS) is a nanomachine deployed by many Gram-negative bacteria as a weapon against eukaryotic hosts or prokaryotic competitors. It assembles into a bacteriophage tail-like structure that can transport effector proteins into the environment or target cells for competitive survival or pathogenesis. T6SS effectors have been identified by a variety of approaches, including knowledge/hypothesis-dependent and discovery-driven approaches. Here, we review and discuss the methods that have been used to identify T6SS effectors and the biological and biochemical functions of known effectors. On the basis of the nature and transport mechanisms of T6SS effectors, we further propose potential strategies that may be applicable to identify new T6SS effectors.

Burkholderia thailandensis uses acyl-homoserine lactone-mediated quorum sensing systems to regulate hundreds of genes. Here we show that cell-cell contact-dependent type VI secretion (T6S) toxin-immunity systems are among those activated by quorum sensing in B. thailandensis. We also demonstrate that T6S is required to constrain proliferation of quorum sensing mutants in colony cocultures of a BtaR1 quorum-sensing signal receptor mutant and its parent. However, the BtaR1 mutant is not constrained by and outcompetes its parent in broth coculture, presumably because no cell contact occurs and there is a metabolic cost associated with quorum sensing gene activation. The increased fitness of the wild type over the BtaR1 mutant during agar surface growth is dependent on an intact T6SS-1 apparatus. Thus, quorum sensing activates B. thailandensis T6SS-1 growth inhibition and this control serves to police and constrain quorum-sensing mutants. This work defines a novel role for T6SSs in intraspecies mutant control. Bacterial cells communicate with each other by using chemical signals. The signals allow cells living in a group to coordinate their behaviors. This cooperation can help all the cells in the group, yet scientists are puzzled about how it could evolve and persist in a population. This is because individual bacteria that essentially cheat and benefit from the cooperation of the rest of the group without contributing their fair share would have a fitness advantage. Now, Majerczyk et al. show that a bacterium called Burkholderia thailandensis, which is commonly found in soil, poisons neighboring freeloaders to prevent such cheating. In the experiments, bacteria with mutations that allow them to ignore the chemical signals that trigger cooperation have an advantage over cooperative bacteria when the two types of bacteria are grown together in a liquid. However, the uncooperative mutants lose their advantage when they are grown on a surface where the cooperative bacteria touch them. To understand why, Majerczyk et al. examined which genes were active in both types of cells in these situations. These experiments showed that chemical signals released by the cooperative bacteria cause them to produce both toxins and proteins that protect against these toxins. This allows the cooperative cells to poison cheaters that they come in contact with, while suffering no ill effects themselves. This allows the cooperative bacteria, via the signaling chemicals, to police cheats and reward only other cooperative bacteria. The next step will be to determine if other types of bacteria also use this policing strategy and to identify more pairs of genes that contribute to protecting the benefits of cooperation.

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. 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.

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-CTEC869 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-CTEC869 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-CTEC869 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.

The initial discovery of killer toxin-secreting brewery strains of Saccharomyces cerevisiae (S. cerevisiae) in the mid-sixties of the last century marked the beginning of intensive research in the yeast virology field. So far, four different S. cerevisiae killer toxins (K28, K1, K2, and Klus), encoded by cytoplasmic inherited double-stranded RNA viruses (dsRNA) of the Totiviridae family, have been identified. Among these, K28 represents the unique example of a yeast viral killer toxin that enters a sensitive cell by receptor-mediated endocytosis to reach its intracellular target(s). This review summarizes and discusses the most recent advances and current knowledge on yeast killer toxin K28, with special emphasis on its endocytosis and intracellular trafficking, pointing towards future directions and open questions in this still timely and fascinating field of killer yeast research.

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. 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.