ABSTRACT
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.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/growth & development , Membrane Proteins/metabolism , Amino Acid Sequence , Coenzymes/metabolism , Contact Inhibition/genetics , Cysteine Synthase/genetics , Cysteine Synthase/metabolism , Enzyme Activation , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Membrane Proteins/chemistry , Molecular Sequence Data , Protein Binding , Sequence AlignmentABSTRACT
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.
Subject(s)
Bacterial Toxins/genetics , Evolution, Molecular , Genetic Variation , Salmonella typhimurium/genetics , Amino Acid Sequence , Animals , Bacterial Toxins/biosynthesis , Cell Proliferation , Gene Expression Regulation, Bacterial , Genetic Fitness , Humans , Mice , Salmonella typhimurium/growth & developmentABSTRACT
Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA 'exoprotein' onto the cell surface. An additional small immunity protein (CdiI) protects CDI(+) cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI(+) cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.
Subject(s)
Bacterial Toxins/metabolism , Uropathogenic Escherichia coli/metabolism , Amino Acid Sequence , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Bacterial Toxins/immunology , Contact Inhibition/immunology , Contact Inhibition/physiology , Enterobacteriaceae/enzymology , Enterobacteriaceae/genetics , Enterobacteriaceae/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Uropathogenic Escherichia coli/enzymology , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/growth & developmentABSTRACT
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.