Antibacterial T6SS effectors with a VRR-Nuc domain are structure-specific nucleases

The type VI secretion system (T6SS) secretes antibacterial effectors into target competitors. <i>Salmonella</i> spp. encode five phylogenetically distinct T6SSs. Here, we characterize the function of the SPI-22 T6SS of <i>Salmonella bongori</i> showing that it has antibacterial activity and identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins with a DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in <i>Escherichia coli</i> and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV3 recognizes specific DNA structures and preferentially cleave splayed arms, generating DNA double-strand breaks and inducing the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of <i>Salmonella</i> pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanisms regulating the activity of these toxins.