Characterization Of Quorum Sensing In Pseudoalteromonas Sp. R3 And Its Regulation Mechanism By SRNA

Quorum sensing(QS)is an important communication system by which bacteria produce,release and detect signal molecules,and successively orchestrate target gene expression in response to changes in cell density and species composition of the community.Pseudoalteromonas are typical marine bacteria broadly found in versatile environments,the diverse bioactive compounds produced by Pseudoalteromonas suggesting it possess important ecological and biological significance in marine environent.They have broad applications in biological control in aquaculture,but current knowledge on their QS systems is extremely fragmentary.Previously,whole genome sequencing of Pseudoalteromonas sp.R3 was performed.In this study,we aim to predictand explore the QS of strain R3 through genome sequence and analysis accompanied with related experiments.In addition,we play an important role inthe mechanism of regulation by sRNA on QS in Pseudoalteromonas.Accordingly,a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3.Among them,the typical LuxI/R-type QS system using acyl-homoserine lactones(AHLs)as signal molecules was characterized.Sequence similarity analysis indicated thatluxIencoding AHLs synthase has 34.9% identity with representativeLuxI enzyme,which meansprobablynovel.TheluxR encoding AHLs receptor is directly adjacent to the luxI downstream.Notably,mutagenesis analysis demonstrated that two mutants lost ability to produce AHLs.The result of qPCR indicated thatLuxI and LuxRpositively affect each other at transcriptional level,and both of them control the synthesis of AHLs.Thin-layer chromatography analysis showed that strain R3mayproduce two types of AHLs,including 3-OH-C6-HSL and C8-HSL.In order to study the effect of sRNA on QS,we constructed amutant strainwith deleted hfq encoding RNA chaperone Hfq.Theanalysis of qPCR showed that Hfqpositively affects the expression level of luxI and luxRat transcriptional level.By using cross-feeding,we found the phenotype of the strain with complementationof luxRwithout UTR regionfails to be restored.In contrast,the complementation of luxIwithout UTR region results inrecovery ofits phenotype,collectively revealing that the luxRbut not the luxIis regulatedby Hfq-involved system through binding to UTR.Accordingly,it was suggested that 5’UTR region of luxRis the regulation area of Hfq-dependent sRNA.Afterwards,through bioinformatics together with analysis of RNA-seq and qPCRwe predicted and identified two candidate Hfq-dependent sRNAs which involve the regulation of luxRin QS.