The Regulation Of RpoN For Biofilm And Related Phenotypes Under Different Density Conditions In Vibrio Alginolyticus HN08155

Vibrio alginolyticus is a major causative agent of vibriosis in both marine animals and human beings.RpoN(δ54)is a global regulator that controls crucial virulence-associated phenotypes.In V.alginolyticus,rpoN enhances flagellation,T6 SS activity and ECP hemolytic activity but suppresses protease and siderophore production.However,the impact of rpoN on the complex regulatory mechanisms of biofilm formation has remained unclear.In the study,we constructed the deletion mutant strain ΔrpoN,the complementary strain ΔrpoN-C and the negative control strain ΔrpoN-Z to examine the correlation between rpoN and biofilm formation in V.alginolyticus HN08155(WT)under different cell density conditions.We confirmed that there was no difference in growth under shock culture conditions among WT,ΔrpoN,ΔrpoN-C and ΔrpoN-Z.However,compared with those of WT and ΔrpoN-C,the growth abilities of ΔrpoN and ΔrpoN-Z decreased and the growing conditions changed,with bacteria concentrating at the gas-liquid interface under static culture conditions.The growth abilities of ΔrpoN and ΔrpoN-Z on plate medium decreased in early cultivation(≤12 h),but they were similar to those of WT and ΔrpoN-C within 36 h.Moreover,deletion of rpoN resulted in feeble motility and lack of flagellum.The colony morphologies of ΔrpoN and ΔrpoN-Z transformed from smooth to rugose.The biofilms of WT and ΔrpoN-C were structured at low cell density(LCD)and disintegrated at high cell density(HCD),while the biofilms of ΔrpoN and ΔrpoN-Z were absent at LCD and accumulated excessive at HCD.Interestingly,there was a spotty pellicle at the gas-liquid interface when ΔrpoN and ΔrpoN-Z were statically cultured after 36 h.In WT,the expression levels of rpoN and hap R were consistent with the trend of its biofilm formation(LCD>HCD).In ΔrpoN,the expression level of hap R also followed the trend of its biofilm formation(HCD>LCD).Nevertheless,there was no remarkable difference in the expression of vps R between WT and ΔrpoN at LCD,while in ΔrpoN,the expression of vps R was double that in WT at HCD.Incredibly,aph A was not observed in HN08155.In conclusion,rpoN initiated the flagellum-dependent biofilm formation(FDBF)mechanism and promoted biofilm formation at LCD.The presence of rpoN inhibited VPS and enabled flagellation,which led to biofilm decomposition at HCD in V.alginoliticus.Although rpoN was not a necessary condition for biofilm formation,it was an essential factor for biofilm decomposition.Specifically,rpoN activated hap R and was not required for the expression of vps R at LCD,while it inhibited the expression of both hap R and vps R at HCD.We speculate that rpoN promoted biofilm formation by upregulating hap R.Moreover,an unrevealed gene rpo S might activate hap R and subsequently promote biofilm formation if rpoN is deletion at HCD.In addition,we established that rpoN is involved in the metabolic utilization of oxygen.