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.

Cellular and Molecular Microbiology Faculté des Sciences Université Libre de Bruxelles 6041 Gosselies Belgium

Centre for Bacterial Cell Biology Biosciences Institute Newcastle University NE2 4AX Newcastle upon Tyne United Kingdom

Department of Molecular Biology Umeå University 901 87 Umeå Sweden

Department of Molecular Biology Umeå University 901 87 Umeå Sweden;

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences CZ 166 10 Prague 6 Czech Republic

Institute of Technology University of Tartu 50411 Tartu Estonia

Laboratory for Molecular Infection Medicine Sweden Umeå University SE 901 87 Umeå Sweden

Walloon Excellence in Life Sciences and Biotechnology 1200 Brussels Belgium

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