PIX is an N-terminal delivery domain that defines a class of polymorphic T6SS effectors in Enterobacterales

The type VI secretion system (T6SS), a widespread protein delivery apparatus, plays a role in bacterial competition by delivering toxic effectors into neighboring cells. Identifying new T6SS effectors and deciphering the mechanism that governs their secretion remain major challenges. Here, we report two orphan antibacterial T6SS effectors in the pathogen Pantoea agglomerans (Pa). These effectors share an N-terminal domain, Pantoea type six (PIX), that defines a widespread class of polymorphic T6SS effectors in Enterobacterales. We show that the PIX domain is necessary and sufficient for T6SS-mediated effector secretion and that PIX binds to a specialized Pa VgrG protein outside its C-terminal toxic domain. Our findings underline the importance of identifying and characterizing delivery domains in polymorphic toxin classes as a tool to reveal effectors and shed light on effector delivery mechanisms.