Study On The Mechanism Of OMVs On The Pseudoalteromonas Marina Biofilm Formation And The Settlement Of Mytilus Coruscus Plantigrades

Mytilus coruscus is one of the common marine benthic animals and also an important economic shellfish,which is widely living in the coastal areas of China.The settlement of plantigrades is an important process of mussels from artificial culture to wild culture.The output of seedlings is directly related to the economic benefits of mussels,and this process can be regulated by biofilm.Therefore,it is of great value and significance to screen and identify environmentally friendly and efficient inducers from biofilms and clarify the mechanism of mussels receiving stimulation.Outer membrane vesicles were widely existed in gram-negative bacteria.They are lipid spherical structures wrapped by phospholipid bilayer and contain a variety of bioactive substances,such as lipopolysaccharide.OMVs participate in many important biological processes,including mediating bacteria-bacteria and bacteria-host interactions,transporting bacterial signal molecules,and maintaining bacterial survival.In addition,OMVs can improve the formation of bacterial biofilm.Hence,this study extracted and purified OMVs from P.marina,and added them to wild-type bacteria to form biofilm by co-incubation,detected the induced effect of the formed biofilm on the settlement of mussel,compared the biofilm forming ability and the changes of extracellular products,then,analyzed the relationship between them and the settlement of mussel plantigrades.The aim is to investigate the influence of OMVs on the bacterial biofilm formation of Pseudoalteromonas marina and the colonization of mussel plantigrades.The OMVs formation defect strain Δtol B was obtained by gene knockout technology to determine whether the tol B mutant would affect the settlement of mussels and change the characteristics of biofilm.The results of this study are as follows:1.OMVs induce the settlement of mussels and improve the biofilm formation ability of P.marina.In this study,OMVs were extracted and purified from P.marina and added to wild-type bacteria to forme biofilms,with the concentration of 0.1,1,10,20 μg/m L.The results show that when added the OMVs at 10 μg/m L,the biofilm had the highest settlement inducing activity to the mussel plantigrades,and the rate of settlement reached 55%.Meanwhile,the bacterial density and biofilms thickness showed an increasing trend,and the extracellular products also increased with the increase of the concentration of OMVs.The content of α-polysaccharide,β-polysaccharide,lipid and protein increased by 115.95%,96.05%,98.74% and 199.54%,respectively.The results of correlation analysis showed that OMVs increased the settlement inducing activity of mussel by regulating the changes of lipid content in the biofilm.2.OMVs induce the settlement of mussels via the LPS.In order to explore the effect of the OMV synthesis regulation gene tol B on the settlement of mussels,and further verify the function of OMVs.In the experiment,homologous recombination was used to obtained the tol B knockout strain,the results of the characteristics of biofilms were compared and analyzed.The deletion of the tol B gene caused a decrease in c-di-GMP regulation,which could lead to a decrease in OMVs,bacterial activity and biofilm formation capacity.To observe the alterations of extracellular substances in wild-type biofilms,Δtol B biofilms and OMV-reinforced biofilms,confocal laser scanning microscopy was utilized.The deletion of tol B gene reduced the content of extracellular active substances in the biofilms.After the addition of OMVs,the α-polysaccharide,β-polysaccharide,lipids and proteins were replenished to different degrees.After phospholipase C treatment of lipopolysaccharide in OMVs,the content of LPS also decreased by 94.87%,and the induced activity of OMV decreased by 61.11%.To sum up,the OMVs extracted from P.marina can directly promote the settlement of mussels,but also indirectly affect the settlement via changing the formation and biological characteristics of biofilms.The tol B gene affects different extracellular products such as polysaccharides,protein and lipids by regulating the function of Tol-Pal system.The material complement of OMVs changes the reduction of induction activity caused by tol B gene deletion,indicating that this change is reversible.The content of LPS suggests that LPS may play an important role in OMVs.The settlement of mussels is regulated by many factors,not a single factor.There may be a stepwise regulation relationship in the information exchange and material transfer with the mussels host.This study provide a theoretical basis for the molecular mechanism of regulation of OMVs on biofilm formation and settlement of mussels,and give a new idea for improving the yield of mussel seedlings and promoting the development of mussel artificial breeding industry.