Multicellular Bacteria Deploy the Type VI Secretion System to Preemptively Strike Neighboring Cells

Abstract

The Type VI Secretion System (T6SS) functions in bacteria as a contractile nanomachine that punctures and delivers lethal effectors to a target cell. Virtually nothing is known about the lifestyle or physiology that dictates when bacteria normally produce their T6SS, which prevents a clear understanding of how bacteria benefit from its action in their natural habitat. Proteus mirabilis undergoes a characteristic developmental process to coordinate a multicellular swarming behavior and will discriminate itself from another Proteus isolate during swarming, resulting in a visible boundary termed a Dienes line. Using transposon mutagenesis, we discovered that this recognition phenomenon requires the lethal action of the T6SS. All mutants identified in the genetic screen had insertions within a single 33.5-kb region that encodes a T6SS and cognate Hcp-VrgG-linked effectors. The identified T6SS and primary effector operons were characterized by killing assays, by construction of additional mutants, by complementation, and by examining the activity of the type VI secretion system in real-time using live-cell microscopy on opposing swarms. We show that lethal T6SS-dependent activity occurs when a dominant strain infiltrates deeply beyond the boundary of the two swarms. Using this multicellular model, we found that social recognition in bacteria, underlying killing, and immunity to killing all require cell-cell contact, can be assigned to specific genes, and are dependent on the T6SS. The ability to survive a lethal T6SS attack equates to “recognition”. In contrast to the current model of T6SS being an offensive or defensive weapon our findings support a preemptive mechanism by which an entire population indiscriminately uses the T6SS for contact-dependent delivery of effectors during its cooperative mode of growth. Bacterial Type VI Secretion Systems (T6SS) function as contractile nanomachines to puncture target cells and deliver lethal effectors. Little is known about the lifestyle or physiology dictating when bacteria normally express their T6SS. Proteus mirabilis undergoes a characteristic developmental process to coordinate multicellular swarming behavior and discriminates itself from other Proteus isolates during swarming, by a visible boundary termed a Dienes line. We report this phenomenon, first noted in 1946, requires the lethal action of the T6SS, T6SS-dependent effectors, and immunity proteins. T6SS-mediated lethality is unique to morphologically distinct swarmer cells, and when it occurs requires direct contact. Using this multicellular system, we report that the pre-formed T6SS strikes neighboring cells upon cell-cell contact. Our findings support a preemptive mechanism by which an entire population indiscriminately uses the T6SS during a cooperative behavior and that social recognition in bacteria is immunity to lethal T6SS attack.