A type VI secretion ADP-ribosyltransferase Rhs toxin selectively modifies charged tRNAThr(GGU) anticodon loops to block ACC codon decoding

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The type VI secretion system (T6SS) is a sophisticated machine that uses a contractile mechanism to deliver antibacterial toxins into neighboring cells, hence participating to interbacterial competition and pathogenesis. 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-NAD 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.