Screen And Indentification Of The Quorum Sensing Transcriptional Regulators In Vibrio Alginolyticus

Vibrio alginolyticus belongs to Gram negative bacteria and is an important opportunistic pathogen causing vibriosis in many commercially important fish species.A better understanding of V.alginolyticus pathogenesis may help to produce a safe and effective vaccine to prevent vibriosis.Many virulence factors have been identified,including quorum sensing system,secretory system,iron uptake system,motility,biofilm and so on.Quorum sensing system,as the most important virulence factor,can regulate other virulence factors.AphA and LuxR are the master regulators of quorum sensing system,regulating a variety of genes.However,the transcriptional regulators of aphA and luxR are poorly understood.In this study,in order to find the transcriptional regulators of aphA and luxR in.alginolyticu V,a bacterial one-hybrid system and the library of V.alginolyticus transcriptional regulators were constructed.And the mechanisms mediated by two of the transcriptional regulators were studied.It lays the foundation of analysis in which quorum sensing system mediating cell density and other physiological signals to regulate genes and showing multicellular group behavior.In this study,we constructed a new bacterial one-hybrid system and the library of V.alginolyticus transcriptional regulators.Putative 99 and 76 transcriptional regulators had been screend from the library binding the promoters of aphA and luxR respectively.Through the comparison and analysis,we found 53 regulators binding with the two promoters.And three proteins of them,including SoxR,MetR and AsnC,were purified.Then EMSA proved SoxR,MetR and AsnC binding on the promoters of luxR and aphA.Furthermore,we constructed seven mutations.HPA and westernblot identified that these seven regulators controlling the expressions of Asp,the virulence protein in V.alginolyticus.AphB activates the expression of exotoxin Asp by binding directly to promoter regions of asp and the quorum sensing master regulator luxR.DNase I footprinting analysis uncovers the distinct AphB binding sites?BBS?in these promoters.BBS in the luxR promoter region overlaps with that of LuxR binding site I.Electrophoretic mobility shift assays?EMSA?indicated the cooperation between AphB and LuxR,which positively control the luxR promoter activity.Here,AphB is found as a key in vivo virulence and biofilm regulator in V.alginolyticus.However,its regulon remains largely unknown.Chromatin immunoprecipitation followed by high-throughput DNA sequencing enabled detection of 20 loci that contain AphB-binding peaks in V.alginolyticus genome.AphB binds directly to the promoter of itself and positively controls its own expression at later growth stages.An AphB-specific binding consensus was identified by EMSA,and the regulation of genes near such binding sites were confirmed by quantitative real time PCR analysis.Taken together,this study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and quorum sensing controlled virulence.The production encoded by EPGS₁8672 gene is a LysR transcriptional regulator.18672 is the co-transcriptional regulator of aphA and luxR.It regulates aphA and luxR by binding their promoters respectively,and feedback controlled by them.18672 also negative regulates itself via binding the palindrome on its promoter.By sterilization experiment and the assay of virulence for zebra fish,the deletion of EPGS₁8672 leads to the lost of ability in killing bacteria and the reduction of virulence.On the basis of LysR family transcriptional regulators responsing to the various signals outside,the quorum sensing expression is regulated by more unknown extracellular signals,such as EPGS₁8672 may response to the contact with other cells,and AphB response to the acid.