VgrG Gene Promotes Mussel Settlement By Regulating Extracellular OMV Through T6SS

Mytilus coruscus have strong reproductive ability and environmental adaptability,and are representative large fouled shellfish in China.Because it is often attached to the bottom of ships and offshore buildings,it causes huge economic losses to the shipping industry and offshore engineering construction industry every year.Biofilms are widely distributed and can induce settlement in marine invertebrates.T6 SS plays an important role in bacterial growth and biofilm formation,but how bacterial T6 SS regulates biofilm formation and affects mussel settlement is unclear,limiting our understanding of the complex physiological and ecological role of T6 SS.As an important protein secretion system,it is unknown whether T6 SS can affect the settlement of M.coruscus by affecting the biological characteristics of the biofilm,or whether it can affect the secretion of substances to affect the settlement of M.coruscus.In this paper,we construct a mutant of the deletion of the core gene vgrG of Leisingera aquaemixtae ECSMC170901 T6 SS to explore the mechanism of action of T6 SS on the settlement of M.coruscus.1: VgrG gene deletion can reduce the induction ability of L.aquaemixtae biofilm on M.coruscus.The valine-glycine repeat protein G,a component of L.aquaemixtae type VI.secretory system and effector protein,synthesized vgrG and supplemented the gene.The characteristics of L.aquaemixtae,ΔvgrG and CvgrG were analyzed.The results showed that the deletion of vgrG gene significantly reduced the growth capacity of L.aquaemixtae,the color of colonies became transparent,the motility was enhanced,the thickness of the biofilm film decreased,the bacterial density decreased.After the vgrG gene was replenished,all the characteristics returned to the same as those of wild-type bacterial.In this experiment,it was found that the protein level in EPS of the mutant biofilm decreased by 68.98% compared with wild-type bacteria.Moreover,the ability of mutant bacteria biofilm to induce the settlement of M.coruscus was significantly reduced.Previous studies have found that the protein in the bacterial biofilm is one of the factors that induce the settlement of M.coruscus,so it is speculated that the decrease in the induced activity of mutant bacteria biofilm is caused by the decrease of content in the bacterial extracellular proteins.2: VgrG protein affects the content of OMV in L.aquaemixtae biofilm to promote the settlement of M.coruscusAs an important protein secretion system of bacteria,T6 SS can not only secrete many effector proteins,but also affect the physiological functions of other protein secretion systems.The vgrG gene affects the protein secretion of L.aquaemixtae and affects the ability of bacterial biofilms to induce M.coruscus,so we investigated the ability of VgrG protein to induce settlement of M.coruscus.Through the VgrG protein induction experiment,it was found that the protein did not induce activity,but the induction of the biofilm formed by the co-culture of mutant bacteria and VgrG protein to the settlement of M.coruscus returned to the level of wild-type bacteria.It was speculated that T6 SS affected the secretion of other substances of bacteria,which enhanced the biofilm induced activity of mutant bacteria.Recent studies have found that soluble proteins as encapsulated periplasm and exoadhesions can bind to OMV and T6 SS can recruit bacteria-secreted OMV to bacterial surfaces.Based on this,the OMV of wild-type bacteria and mutant bacteria was extracted and quantified,and it was found that the deletion of vgrG gene led to a significant decrease in the OMV content of bacteria biofilm.OMV extracted from the biofilm of wild-type bacterial can induce the settlement of M.coruscus.It is hypothesized that VgrG affects the induce ability of biofilm on the settlement of M.coruscus by affecting T6 SS to regulate the extracellular OMV content of bacteria.