Summary paragraphBacteria have developed mechanisms to communicate and compete with one another in diverse environments 1. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli 2. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional immunity protein (CdiI) protects CDI+ cells from autoinhibition 2, 3. The mechanisms by which CDI blocks cell growth and 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 utilized 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 in both laboratory media and upon a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network that plays an important role in bacterial competition.

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.

Cells employ many mechanisms to ensure quality control during protein biosynthesis. Here, we show that, during the pausing of a bacterial ribosome, the mRNA being translated is cleaved at a site within or immediately adjacent to the A site codon. The extent of this A site mRNA cleavage is correlated with the extent of ribosome pausing as assayed by tmRNA-mediated tagging of the nascent polypeptide. Cleavage does not require tmRNA, the ribosomal alarmone (p)ppGpp, or bacterial toxins such as RelE which have been shown to stimulate a similar activity. Translation is required for cleavage, suggesting that the ribosome participates in the reaction in some fashion. When normal protein synthesis is compromised, A site mRNA cleavage and the tmRNA system provide a mechanism for reducing translational errors and the production of aberrant and potentially harmful polypeptides.

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.

Summary Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.