The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner.ImportanceThe type six secretion system (T6SS) is a molecular poison-tipped spear that bacteria use to kill nearby competitors. To prevent self killing, they use antitoxins called immunity genes that specifically neutralize the poisons. Beyond immunity genes, multiple additional defences have recently been discovered but there are likely many more across the genomes of diverse species. To help discover these novel mechanisms, we designed a high-throughput method that can be used in numerous different species to rapidly identify genes involved in sensitivity to T6SS attacks. Using T6SS ‘killers’ delivering individual poisons and two commonly studied ‘prey’ bacteria, we show proof-of-principle that the technique can discover proteins that make the prey cells more resistant or sensitive to particular poisons. This will greatly improve the speed at which genes influencing the T6SS can be identified and selected for further study in follow-up analyses.
