Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin−antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS–antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

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.

22Under stressful conditions, bacterial RelA-SpoT Homologue (RSH) enzymes synthesise the alarmone 23 (p)ppGpp, a nucleotide messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope 24 with stress, and at high concentrations inhibits the process of protein synthesis and bacterial growth 25 to save and redirect resources until conditions improve. Single domain Small Alarmone Synthetases 26 (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the 27 hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA and 28SpoT. We have discovered that multiple SAS subfamilies can be encoded in broadly distributed 29 conserved bicistronic operon architectures in bacteria and bacteriophages that are reminiscent of 30 those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being 31 toxic (toxSASs), with neutralisation by the protein products of six neighbouring antitoxin genes. The 32 toxicity of Cellulomonas marina ToxSAS FaRel is mediated by alarmone accumulation combined with 33 depletion of cellular ATP and GTP pools, and this is counteracted by its HD domain-containing 34 antitoxin. Thus, the ToxSAS-antiToxSAS system is a novel TA paradigm comprising multiple different 35 antitoxins that exemplifies how ancient nucleotide-based signalling mechanisms can be repurposed 36 as TA modules during evolution, potentially multiple times independently. 37 1 Bacteria encounter a variety of different environmental conditions during their life cycles, to which 2 they need to respond and adapt in order to survive. This can include slowing down their growth and 3 redirecting their metabolic resources during nutritional stress, until conditions improve and the 4 growth rate can increase. One of the main signals that bacteria use for signalling stress are the 5 alarmone nucleotides ppGpp and pppGpp, collectively referred to as (p)ppGpp 1 . At high 6 concentrations (p)ppGpp is a potent inhibitor of bacterial growth 2 , targeting transcription, 7 translation and ribosome assembly 1 . (p)ppGpp is produced and degraded by proteins of the 8 RelA/SpoT homologue (RSH) superfamily, named after the two Escherichia coli representatives -9 multi-domain 'long' RSH factors RelA and SpoT 3 . In addition to long RSHs, bacteria can encode single-10 domain RSHs: Small Alarmone Synthetases (SAS) and Small Alarmone Hydrolases (SAH). 11It is currently unknown why some bacteria carry multiple SASs and SAHs, which can belong to many 12 different subfamilies. Conservation of gene order through evolution can reveal potentially interacting 13 proteins and shed light on the cellular role of proteins 4 . Therefore, we developed a computational 14 tool -FlaGs, standing for Flanking Genes 5 -for analysing the conservation of genomic 15 neighbourhoods, and applied it to our updated database of RSH sequences classified into 16 subfamilies. Surprisingly, we find that some subfamilies of SAS can be encoded in apparently bi-(and 17 sometimes tri-) ...

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.

Bacteria have evolved sophisticated and diverse immunity mechanisms to protect themselves against a nearly constant onslaught of bacteriophages. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns (PAMPs), many bacterial immune systems that respond to bacteriophage infection require a phage-specific trigger to be activated. However, the identities of such triggers and the mechanistic basis of sensing remain almost completely unknown. Here, we discover and investigate the anti-phage function of a fused toxin-antitoxin (TA) system called CapRelSJ46 that protects E. coli against diverse phages. Through genetic, biochemical, and structural analysis, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both an antitoxin element and a phage-infection sensor. Following infection by certain phages, the newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to then 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 TA-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a Red Queen conflict, revealing a new front in the intense coevolutionary battle being waged by phage and bacteria. With capsid proteins of some eukaryotic viruses known to stimulate innate immune signaling in mammalian hosts, our results now reveal an ancient, deeply conserved facet of immunity.