Mussel settlement mediated by bacterial VgrG proteins via extracellular outer membrane vesicles

T6SS plays an important role in biofilm formation, but how bacterial T6SS regulates biofilm formation and its effect on marine invertebrate settlement is still unclear. Here, the role of T6SS spike protein VgrG and VgrG protein synthesis gene on biofilm formation and Mytilus coruscus settlement was examined. Deletion of the core gene vgrG of T6SS resulted in a decrease in the growth capability of Leisingera aquaemixtae and inhibition of the ability of biofilm formation. The OMV content and inducing activity of Delta vgrG biofilms were decreased respec-tively by 20.72% and 59.70% compared with the wild-type biofilms. OMVs induced the settlement of M. coruscus, while VgrG proteins did not induce mussel settlement directly. Addition of VgrG proteins resulted in the upregulation of OMVs in the Delta vgrG biofilms. Adding OMVs caused no increasing inducing activity of Delta vgrG biofilms. Interestingly, the inducing activity of Delta vgrG biofilms both by adding VgrG proteins alone, and by adding VgrG proteins and OMVs in combination increased significantly, and restored to the inducing capability of wild-type biofilms. Thus, bacterial T6SS spike protein VgrG could mediate mussel settlement by increasing the OMV content of biofilms. The present findings shed new light on the interaction between T6SS and marine invertebrate settlement.