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

Abstract

Abstract 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.