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Here, we characterize the T6SS-associated rearrangement hotspot RhsF polymorphic toxin from Chromobacterium violaceum. Toxicity assays showed that the RhsF C-terminal extension, TreThr, is a cytoplasmic-acting antibacterial toxin, which is neutralized by the downstream immunity protein. Structural modeling and mutagenesis demonstrated that TreThr belongs to the ADP-ribosyltransferase (ART) family, adopting a canonical ART fold. Using coupled in vitro transcription-translation assays, we found that TreThr inhibits protein synthesis in a Nicotinamide Adenine Dinucleotide (NAD)-dependent manner, through isoacceptor-specific ADP-ribosylation of transfer RNAs (tRNAs). Biotin\u2013NAD labeling experiments and mapping further revealed that TreThr modifies the GGU anticodon of charged thrT and thrV tRNA isoacceptors and prevents decoding of threonine ACC codons.<\/jats:p>","DOI":"10.1093\/nar\/gkag922","type":"journal-article","created":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T11:44:18Z","timestamp":1789213458000},"source":"Crossref","is-referenced-by-count":0,"title":["A type VI secretion ADP-ribosyltransferase Rhs toxin selectively modifies charged tRNAThr(GGU) anticodon loops to block ACC codon decoding"],"prefix":"10.1093","volume":"54","author":[{"given":"Ma\u00efal\u00e8ne","family":"Chabalier","sequence":"first","affiliation":[{"name":"Laboratoire d\u2019Ing\u00e9nierie des Syst\u00e8mes Macromol\u00e9culaires (LISM, UMR7255), Aix-Marseille Universit\u00e9, CNRS , 31 Chemin Joseph Aiguier CS7071, Marseille Cedex 20 13402 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonas B","family":"Desjardins","sequence":"additional","affiliation":[{"name":"Laboratoire d\u2019Ing\u00e9nierie des Syst\u00e8mes Macromol\u00e9culaires (LISM, UMR7255), Aix-Marseille Universit\u00e9, CNRS , 31 Chemin Joseph Aiguier CS7071, Marseille Cedex 20 13402 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julius","family":"Martinkus","sequence":"additional","affiliation":[{"name":"Laboratoire d\u2019Ing\u00e9nierie des Syst\u00e8mes Macromol\u00e9culaires (LISM, UMR7255), Aix-Marseille Universit\u00e9, CNRS , 31 Chemin Joseph Aiguier CS7071, Marseille Cedex 20 13402 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0611-9179","authenticated-orcid":false,"given":"Eric","family":"Cascales","sequence":"additional","affiliation":[{"name":"Laboratoire d\u2019Ing\u00e9nierie des Syst\u00e8mes Macromol\u00e9culaires (LISM, UMR7255), Aix-Marseille Universit\u00e9, CNRS , 31 Chemin Joseph Aiguier CS7071, Marseille Cedex 20 13402 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"key":"2026092805050240100_B1","doi-asserted-by":"publisher","first-page":"e299","DOI":"10.1371\/journal.pbio.0040299","article-title":"Cooperation among microorganisms","volume":"4","author":"Wingreen","year":"2006","journal-title":"PLoS Biol"},{"key":"2026092805050240100_B2","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1038\/nrmicro2259","article-title":"Bacterial competition: surviving and thriving in the microbial jungle","volume":"8","author":"Hibbing","year":"2010","journal-title":"Nat Rev Micro"},{"key":"2026092805050240100_B3","doi-asserted-by":"publisher","first-page":"663","DOI":"10.1126\/science.aad2602","article-title":"The ecology of the microbiome: networks, competition, and 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