Expression Of Quorum Sensing System Gene HalI/HalR And Its Inhibitors In Hafnia Alvei Isolated From Turbot

With the innovation and development of marine culture in greenhouse,the marine production of turbot increase year after year,meanwhile,with the rapid development of fresh food cold chain technology,cold chain logistics and supermarket retail industry,consumption market of fresh and clean fish and fillet of turbot is expanding.However,microorganisms are the most common cause of food spoilage,and some studies have observed that spoilage characteristics of microorganisms is closely related to quorum sensing（QS）.And most studies focus on phenotype of QS,there was limited information about genes regulated by QS in Hafnia alvei（H.alvei）.In this context,H.alvei isolated from spoiled turbot was studied in this paper,and the halI/halR gene regulated by QS was screened by molecular biological methods.The influence of different environmental factors on expression of genes regulated by QS were treated,and the exogenous signal molecules were added to explore differences in genes expression and utilization ability of H.alvei.Furthermore,Chromobacterium violaceum（CV026）was used to detect the QS inhibitory activity of vanillin,and further explore the inhibitory mechanism at the molecular level by the method of real-time quantitative PCR（RTq-PCR）and molecular docking.Finally,the quorum sensing inhibitor vanillin was used to verify the preservation effect of refrigerated turbot fillets.This paper aims to explore the QS mechanism of spoilage Hafnia alvei and the inhibition mechanism of vanillin,and provide a theoretical basis for the establishment of aquatic product preservation technology based on the QS system of the spoilage microorganism.1 The genes（halI/halR）regulated by QS in H.alvei were screened and verified by the method of molecular biology,such as Blast and nucleic acid gel electrophoresis.2 The effect of different environmental factors and different AHLs on the expression of halI/halR gene regulated by QS Hafnia alvei using RTq-PCR,and 16S rRNA was used as internal reference,according to the calculation of??Ct.The results suggested that the expression of halI was in the order of xylose,fructose,glucose,maltose,lactose and sucrose,and the expression of halR was xylose,fructose,sucrose,glucose,lactose and maltose from large to small in the different carbon sources.Based on these results,it can be concluded that the addition of xylose could significantly up-regulate the expression of halI and halR genes.Moreover,the expression of halI and halR genes were inhibited under alkaline conditions（pH>7）,and the same results were observed when exceeding optimum temperature（28℃）.Furthermore,the results of RTq-PCR indicated the signal molecules present in the environment could be utilized by Hafnia alvei,and stimulate genes expression,especially C₆-HSL.Finally,based on the results of molecular docking,there is a potential mechanism that the stronger the binding ability between AHLs and the HalR could stimulate halR gene expression.3 We investigated the effect of vanillin as quorum sensing inhibitor using the CV026 as a biosensor strain,and further explored the potential inhibitory mechanism.The results indicated that the violacein production reduced significantly at the sub-minimal inhibitory concentration（sub-MIC）of vanillin,and the AHLs secretion was only was 0.535 and0.874μg/mL for C₆-HSLand C₈-HSL,at the 1.0 mg/mL of vanillin.Then the biofilm structure of Hafnia alvei was observed by optical microscopy and scanning electron microscopy（SEM）,the biofilm structure became loose after treated with different sub-MIC of vanillin.Finally,we explore the inhibitory mechanism of vanillin as quorum sensing inhibitor（QSI）at the molecular level using the method of RTq-PCR and molecular docking.The results showed that vanillin and C₆-HSL have the same site in HalR,suggesting potential competitive tendency between them.4 The effect of quorum sensing inhibitor vanillin on turbot fillets during storage at 4℃was studied.The results were revealed the TVB-N value significantly reduced,and the pH fluctuation range is small,after vanillin treatment,however,there was no significant difference between vanillin treatment and the control for total viable counts,based on these results,the shelf life of refrigerated turbot fillets can be extended for 2-3days after vanillin treatment.