Abstract
RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.
Highlights
Toxin-antitoxin (TA) systems are a class of highly diverse and widespread small operons found in bacterial, archaeal, and bacteriophage genomes (Fraikin et al, 2020; Harms et al, 2018)
Representatives of FaRel2, PhRel, PhRel2, and CapRel toxSAS subfamilies inhibit protein synthesis the mechanism of toxicity used by PhRel, PhRel2, FaRel2, and CapRel toxSAS subfamilies is as yet uncharacterized, we initially assumed that these toxSASs, just as C. marina FaRel TA toxin (Jimmy et al, 2020) and the Tas1 toxic RelA-SpoT Homolog (RSH) effector of Pseudomonas aeruginosa Type VI secretion system (Ahmad et al, 2019), inhibit bacterial growth by producing pApp
As we have shown earlier, the FLAG3 tag does not interfere with the toxicity of FaRel2 or the ability of the antitoxin to counteract the protein (Jimmy et al, 2020)
Summary
Toxin-antitoxin (TA) systems are a class of highly diverse and widespread small operons found in bacterial, archaeal, and bacteriophage genomes (Fraikin et al, 2020; Harms et al, 2018). TAs have a broad range of functions, including bacterial defense against bacteriophages, phage competition for infection of bacteria, and stabilization of transposons, plasmids, and bacterial genomes, all of which rely on the highly potent toxicity of the protein toxin controlled by the protein- or RNA-based antitoxin (Blower et al, 2009; Fiedoruk et al, 2015; Guegler and Laub, 2021; Jaffeet al., 1985; Lima-Mendez et al, 2020; Song and Wood, 2020). The RelA-SpoT Homolog (RSH) protein family of housekeeping stress-response enzymes was only recently recognized to contain TA toxins (Jimmy et al, 2020).
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