Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1–3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin–antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin–antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a ‘Red Queen conflict’5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6–10, our results reveal a deeply conserved facet of immunity.

Bifunctional Rel stringent factors, the most abundant class of RSHs (RelA/SpoT Homologues), are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 of GTP/GDP to synthesize the bacterial alarmone (p)ppGpp, and also catalyse the 3 pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide switch mechanism controls catalysis by T. thermophilus Rel (Rel Tt ). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel Tt (Rel Tt NTD ) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation. Fondation Van Buren to A.G.-P.; the Molecular Infection Medicine Sweden (MIMS), Swedish Research council (grant 2017-03783), and Ragnar Söderberg foundation fellowship to V.H.; J. Hendrix and J. Hofkens are grateful to the Research Foundation Flanders (FWO Vlaanderen, G0B4915N) and large infrastructure grant ZW15_09 GOH6316N) and the KU Leuven Research Fund (C14/16/053); J. Hofkens thanks financial support of the Flemish government through long term structural funding Methusalem (CASAS2, Meth/15/04).

Chromosomal toxin-antitoxin (TA) systems are widespread among free-living bacteria and are supposedly involved in stress tolerance. Here, we report the first TA system identified in the soil bacterium Pseudomonas putida. The system, encoded by the loci PP1586-PP1585, is conserved in pseudomonads and belongs to the HigBA family. The new TA pair was named GraTA for the growth rate-affecting ability of GraT and the antidote activity of GraA. The GraTA system shares many features common to previously described type II TA systems. The overexpression of GraT is toxic to the antitoxin deletion mutants, since the toxin's neutralization is achieved by binding of the antitoxin. Also, the graTA operon structure and autoregulation by antitoxin resemble those of other TA loci. However, we were able to delete the antitoxin gene from the chromosome, which shows the unusually mild toxicity of innate GraT compared to previously described toxins. Furthermore, GraT is a temperature-dependent toxin, as its growth-regulating effect becomes more evident at lower temperatures. Besides affecting the growth rate, GraT also increases membrane permeability, resulting in higher sensitivity to some chemicals, e.g., NaCl and paraquat. Nevertheless, the active toxin helps the bacteria survive under different stressful conditions and increases their tolerance to several antibiotics, including streptomycin, kanamycin, and ciprofloxacin. Therefore, our data suggest that GraT may represent a new class of mild chromosomal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth and metabolism regulation than is possible with strong growth-arresting or bactericidal toxins.

Bacterial toxin-antitoxin (TA) modules are tightly regulated to maintain growth in favorable conditions or growth arrest during stress. A typical regulatory strategy involves the antitoxin binding and repressing its own promoter while the toxin often acts as a co-repressor. Here we show that Pseudomonas putida graTA -encoded antitoxin GraA and toxin GraT differ from other TA proteins in the sense that not the antitoxin but the toxin possesses a flexible region. GraA auto-represses the graTA promoter: two GraA dimers bind cooperatively at opposite sides of the operator sequence. Contrary to other TA modules, GraT is a de-repressor of the graTA promoter as its N-terminal disordered segment prevents the binding of the GraT 2 A 2 complex to the operator. Removal of this region restores operator binding and abrogates Gr aT toxicity. GraTA represents a TA module where a flexible region in the toxin rather than in the antitoxin controls operon expression and toxin activity.

Highlights d Several families of toxSAS RSH toxins specifically inhibit protein synthesis d Translation-inhibiting toxSASs transfer pyrophosphate from ATP to the tRNA 3 0 CCA d 3 0 CCA pyrophosphorylation abrogates tRNA aminoacylation d Some SAH RSH enzymes can reverse the pyrophosphorylation of tRNA