Global identification of Chromobacterium violaceum T6SS effectors reveals an Rhs antibacterial toxin featuring FIX and ADP-ribosyltransferase domains

Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently reported that the environmental bacterial pathogen Chromobacterium violaceum has an active type VI secretion system (T6SS), which plays a crucial role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and was shown to intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented in the presence of RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 [A] resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays confirmed that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal the effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.