Effects Of Capsular Polysaccharide Synthesizing Gene CapC On Biofilm Formation And Settlement And Metamorphosis Of Mytilus Coruscus Of Pseudoalteromonas Marina

Mytilus coruscus is a typical marine macrofouling organism.It is of great ecological and economic significance to study its settlement and metamorphosis mechanism for the prevention of marine macrofouling organisms.Pseudoalteromonas is widespread in the ocean.Most strains can forming biofilms to directionally induce the settlement and metamorphosis of marine invertebrate larvae.According to our previous studies on the biofilm induced metamorphosis of mussel larvae and settlement of juveniles,the results show that the extracellular products of the biofilm,such as polysaccharides,proteins,lipids,etc.,have a certain induction effect on the metamorphosis of mussel larvae and settlement of juveniles,and the extracellular polysaccharides(EPS),such as alginate,colanic acid(CA),and cellulose,have different effects on the biofilm formation and larval metamorphosis.At the same time,the deletion of LPS synthesis gene will lead to the significantly reduce the induction effect of the biofilm on the settlement of juveniles.Previous studies have shown that capsular polysaccharide(CPS)can enhance the aggregation of bacteria on the biofilm by promoting the adhesion between bacterial cells and also between bacteria and matrix.However,it is still unclear whether the CPS has the same effect in Pseudoalteromonas marina,and what molecular mechanism does the CPS affect the formation of the biofilm of P.marina and whether it has an effect on the metamorphosis of mussel larvae and settlement of juveniles.In this paper,the effects of capsular polysaccharide on P.marina biofilm,larval settlement and metamorphosis and juvenile shellfish settlement were discussed by constructing capC deletion strain,which regulated the synthesis of capsular polysaccharide.The conclusions are draw as followed:1.The deletion of capC gene,which regulates the synthesis of CPS,leads to the content of extracellular polysaccharide on the biofilm decreased significantly,which reduced the induction activity of larval metamorphosis and the settlement of juvenile mussel.In order to study the effect of CPS synthesis gene on the formation of bacterial biofilm,the ΔcapC strain was constructed and compared with P.marina strain.It was found that the deletion of capC gene did not make the phenotype of P.marina strain changed significantly.When comparing the growth curves of the two strains,it was found that the growth of the strain was not affected after entering the stationary phase;the swimming motility of ΔcapC did not change significantly,but the production capacity of CPS was decreaed by 38.07%,and the level of cyclic diguanosine monophosphate(c-di-GMP)was 1.94 times higher than before.The analysis of biofilm showed that the bacterial density and biofilm thickness did not change significantly after the deletion of capC gene,but compared with wild-type P.marina strain,theα-polysaccharides on biofilm decreased by 35.93%,the β-polysaccharides on biofilm decreased by 36.85%.By spectrophotometry,it was found that the content of colonic acid(CA)and alginate in biofilm decreased by 20.55% and 22.25%,respectively,while the content of lipopolysaccharide did not show significant change.And compared with wild-type strain,the inducing activity of ΔcapC biofilm to mussel larvae and juveniles decreased significantly.Therefore,the regulation gene of CPS synthesis,capC,can regulate c-di-GMP level through the synthesis of CPS,and affect the content of extracellular products of biofilm and the settlement and metamorphosis of mussel larvae and settlement of juveniles.2.Effect of CPS on biofilm formation of ΔcapC strain and settlement of mussel juveniles.It has been speculated that EPS of P.marina has an effect on the settlement and metamorphosis of M.coruscus larvae.In this study,EPS and CPS of P.marina were extracted respectively,and the inducing activities of both on the secondary settlement of M.coruscus were tested.The results showed that the influence of EPS on settlement of juvenile shellfish had a threshold value,and the overall trend was first increased and then decreased.The effect of CPS on secondary settlement of juveniles with M.coruscus was lower than that of EPS.However,when the concentration of CPS solution reached 50 mg/L,the effect was not significantly different from that when the concentration was 0.5 mg/L.In order to explore the molecular mechanism of CPS in biofilm formation and settlement of juveniles,CPS extracted from strain P.marina was added in the process of forming biofilm of ΔcapC,and the biological characteristics of biofilm were studied.The results showed that when the concentration of CPS solution reached 0.5 mg/L in the process of forming the biofilm of ΔcapC,the ability of biofilm to induce the settlement of mussel juveniles returned to the level of wild-type bacteria,and the biomass of EPS also returned to the same level as that of wild-type bacteria.At the same time,according to the quantitative analysis,the deletion of this gene led to a reduction in the content of CA on the biofilm.When the concentration of 0.5 mg/L CPS solution was supplemented,the content of CA on the biofilm recovered to the same level as that of wild-type bacteria.Therefore,it is speculated that the CPS can promote the increase of the CA of the biofilm,and improve the ability of the biofilm to induce the settlement of juveniles.To sum up,the amount of CPS synthesis of capC mutant strain decreased,the level of c-di GMP increased,and the content of EPS decreased,and the induction of larval metamorphosis and secondary settlement of juvenile mussels significantly decreased.The addition of CPS extracted from P.marina strain can restore the production capacity of extracellular polysaccharide of the mutant strain and its induction capacity can also be restored to the level of wild-type bacteria,indicating that the CPS can induce the settlement of juveniles by promoting the production of the biofilm CA.