Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VI Permeabilizing Effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.