An ART-fold Rhs toxin from Pluralibacter gergoviae defines Tne5, a new clade of NAD(P)⁺ glycohydrolases effectors

The type VI secretion system (T6SS) ia a widespread bacterial nanomachine that mediates interbacterial competition by delivering toxic effectors into neighboring cells. Among these, enzymes targeting nicotinamide adenine dinucleotide cofactors (NAD and NADP) are particularly potent because they rapidly disrupt redox homeostasis and central metabolism. Several families of T6SS-associated NAD(P)-consuming effectors (Tne1-Tne4) have been described. Here, we characterize a T6SS-associated Rhs toxin from Pluralibacter gergoviae. Competition assays show that P. gergoviae kills Escherichia coli in a T6SS-dependent manner. Heterologous production reveals that the Rhs C-terminal extension is toxic in the E. coli cytoplasm and that co-production with the protein encoded downstream neutralizes this activity. AlphaFold3 modeling predicts that the toxin adopts an ADP-ribosyltransferase (ART)-like /{beta} fold with a putative catalytic pocket accommodating NAD. By contrast to T6SS ART toxins described so far, the toxin does not inhibit transcription, translation or cell division, but instead depletes NAD and NADP. Phylogenetic analyses and structural modeling show that this effector defines a new ART-related family of NAD(P) glycohydrolases, which we propose to name Tne5, broadly distributed across antagonistic systems.