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.

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-) ...