A Prelimilary Research On Quorum Sensing And Regulation Of Vibrio Paraheomolyticus

In this paper, quorum sensing(QS) of Vibrio paraheomolyticus VIB461isolatedfrom Macrobrachium rosenbergii was investigated. The effects of quorum sensinginhibitor brominated furanone on VIB461quorum sensing system and relatedcharacterizations were also investigated.This study included the following three parts:1. Detection of quorum sensing signal molecules in Vibrio paraheomolyticusThe AHLs (acyl-homoserine lactones) produced by Vibrio paraheomolyticusVIB461were detected by reporter strains Agrobacterium tumefaciens A136/Chromobacterium violaceum CV026and reverse C18thin layer chromatography. Twodifferent kinds of AHLs were identified as C6-HSL and C8-HSL. The AHLs activitiesof VIB461increased firstly, reached highest level in last log growth phase anddecreased afterwards. Furthermore, the presence of AI-2(autoinducer-2) andCAI-1(cholerae autoinducer-l) were also detected by Vibrio harveyi JMH597andJAF375. The results showed that both AI-2and CAI-1activities were occurred inVIB461. The level of Al-2increased rapidly at the log phase of growth, and kept ahigh level till stationary phase, while CAI-1just showed strong activity from late logphase to early stationary phase.2. Research on LuxS/AI-2-mediated quorum sensing in Vibrio paraheomolyticusThe luxS gene of Vibrio paraheomolyticus VIB461was amplified by PCR and themain coding sequence of388bp was obtained. The luxS gene of VIB461showed amaximum similarity of85%to that of Vibrio harveyi through BLAST.The similaritybetween luxS of VIB461and that of other bacteria decreased gradualy with theestrangement of relationship between them. Expression of luxS under different growthphases and different growth conditions were detected by Real-time PCR, the resultsshowed that the expression of luxS was correlated with AI-2activity. They were bothgrowth phase-dependent and modulated by growth conditions. The luxS transcribed rapidly in log growth phase, and kept a high level till stationary phase. Both luxSexpression and AI-2activity were influenced by different temperature, pH and salinity.The level of luxS expression and AI-2activity both increased under conditionsdeviated from the optimum growth condition but reduced under high temperature.3. Regulation of quorum sensing and QS-related characterizations in Vibrioparaheomolyticus by quorum sensing inhibitorThe inhibitory effect and effective concentration range of the synthetic quorumsensing inhibitor brominated furanone on AI-2quorum sensing activity were detectedby reporter strain Vibrio harveyi JMH597. The result showed that thebioluminenscence of JMH597was inhibited by brominated furanone at concentrationof2mg/L, which indicated that the brominated furanone had strong inhibitory activityon quorum sensing. The minimum inhibitory concentration of brominated furanone toVibrio parahaemolyticus VIB461was20mg/L. The subinhibitory concentrations,5mg/L and10mg/L, were used to determine the regulatory effect of brominatedfuranone on VIB461LuxS/AI-2-mediated quorum sensing and QS-related biofilmand extracellular enzymes. The results showed that brominated furanone couldstrongly inhibit VIB461luxS expression and AI-2activity, which could reduce themby more than80%at a concentration of10mg/L. Brominated furanones couldeffectively inhibit VIB461biofilm formation, but only have slightly inhibitory effecton gelatinase and lecithinase activity, we speculated that these characterizations werenot only regulated by LuxS/AI-2quorum sensing system but also affected by otherfactors.