Programmed Secretion Arrest and Receptor-Triggered Toxin Export during Antibacterial Contact-Dependent Growth Inhibition.

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.

Polymorphic Toxins and Their Immunity Proteins: Diversity, Evolution, and Mechanisms of Delivery.

All bacteria must compete for growth niches and other limited environmental resources. These existential battles are waged at several levels, but one common strategy entails the transfer of growth-inhibitory protein toxins between competing cells. These antibacterial effectors are invariably encoded with immunity proteins that protect cells from intoxication by neighboring siblings. Several effector classes have been described, each designed to breach the cell envelope of target bacteria. Although effector architectures and export pathways tend to be clade specific, phylogenetically distant species often deploy closely related toxin domains. Thus, diverse competition systems are linked through a common reservoir of toxin-immunity pairs that is shared via horizontal gene transfer. These toxin-immunity protein pairs are extraordinarily diverse in sequence, and this polymorphism underpins an important mechanism of self/nonself discrimination in bacteria. This review focuses on the structures, functions, and delivery mechanisms of polymorphic toxin effectors that mediate bacterial competition.

Paradoxical Activation of a Type VI Secretion System Phospholipase Effector by Its Cognate Immunity Protein.

Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG ß-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.

The ß-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion.

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 ß-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 ß-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 ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact ß-cage. Although ∆rhsA ∆rhsB 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 ß-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 ß-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors.

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.

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI).

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.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

Functional plasticity of antibacterial EndoU toxins.

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.

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs.

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 ß-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 CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily.

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.

CDI Systems Are Stably Maintained by a Cell-Contact Mediated Surveillance Mechanism.

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.

Unraveling the essential role of CysK in CDI toxin activation.

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.

Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016).

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

Bacterial contact-dependent delivery systems.

Bacteria have developed remarkable systems that sense neighboring target cells upon contact and initiate a series of events that enhance their survival and growth at the expense of the target cells. Four main classes of bacterial cell surface structures have been identified that interact with prokaryotic or eukaryotic target cells to deliver DNA or protein effectors. Type III secretion systems (T3SS) use a flagellum-like tube to deliver protein effectors into eukaryotic host cells, whereas Type IV systems use a pilus-based system to mediate DNA or protein transfer into recipient cells. The contact-dependent growth inhibition system (CDI) is a Type V system, using a long ß-helical cell surface protein to contact receptors in target cells and deliver a growth inhibitory signal. Type VI systems utilize a phage-like tube and cell puncturing device to secrete effector proteins into both eukaryotic and prokaryotic target cells.

CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria.

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.

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways.

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.

Functional Diversity of Cytotoxic tRNase/Immunity Protein Complexes from Burkholderia pseudomallei.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDI(+) bacteria deploy large CdiA effector proteins, which carry variable C-terminal toxin domains (CdiA-CT). CDI(+) cells also produce CdiI immunity proteins that specifically neutralize cognate CdiA-CT toxins to prevent auto-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(E479) toxin/immunity protein complex from Burkholderia pseudomallei isolate E479. The CdiA-CT(E479) tRNase domain contains a core α/ß-fold that is characteristic of PD(D/E)XK superfamily nucleases. Unexpectedly, the closest structural homolog of CdiA-CT(E479) is another CDI toxin domain from B. pseudomallei 1026b. Although unrelated in sequence, the two B. pseudomallei nuclease domains share similar folds and active-site architectures. By contrast, the CdiI(E479) and CdiI(1026b) immunity proteins share no significant sequence or structural homology. CdiA-CT(E479) and 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.

Selection of orphan Rhs toxin expression in evolved Salmonella enterica serovar Typhimurium.

Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic "orphan" toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the "main" rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein.

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.