Study On The Regulation Mechanism Of A LysR Transcriptional Regulator In Burkholderia Cenocepacia

The quorum sensing（QS）system is a cell density-dependent regulation mechanism used by bacterial cells.It is widely distributed in various pathogenic bacteria and regulates the production of multiple virulence factors.QS system can be used as a target for developing new drugs and has a good application prospect.Burkholderia cenocepacia is a human opportunistic pathogen which usually causes infections in susceptible individuals,such as immunodeficiency patients and cystic fibrosis patients.B.cenocepacia can cause lung infection and deterioration,and even cause fatal pneumonia in severe cases.Previous studies showed that B.cenocepacia mostly employs two types of QS systems,including the N-acylhomoserine lactone（AHL）and cis-2-dodecenoic acid（BDSF）systems,which are involved in regulating a variety of biological functions including biofilm formation,motility,protease production,and virulence,but the downstream regulation mechanism is still unclear.In order to comprehensively explore the QS system regulation network of B.cenocepacia,a novel LysR family transcriptional regulator NlfR（Novel LysR family regulator）was screened out by constructing a Tn5 random insertion mutant library.Preliminary studies have shown that the mutant has a similar phenotype to the QS system mutant.Studies have shown that the LysR family transcriptional regulators are very vital transcriptional regulatory proteins which are involved in regulating a variety of biological functions including secondary metabolism,virulence factors production and the generation of quorum sensing signals.Therefore,we have conducted an in-depth study on the function of NlfR protein and its interaction with the QS system.The results are as follows:We demonstrated here that a LysR family transcriptional regulator NlfR controls biofilm formation and protease production in B.cenocepacia H111.In the nlfR deletion mutant strain,the biofilm and protease production were significantly down-regulated compared to the wild type strain.Electrophoretic mobility shift assay showed that NlfR directly binds to the promoter sequence of the gene cluster bclACB,and then regulates the expression of the bclACB gene cluster,suggesting that NlfR directly binds to the promoter of target gene to directly regulate its expression.We performed RT-qPCR to detect the expression of nlfR inΔrpfF_BC,ΔrpfR,ΔcepI,ΔcepR and wild-type H111 strains,the results showed that the expression of nlfR was significantly down-regulated compared to that in the wild-type strain.Theβ-galactosidase activity assays also revealed that the nlfR expression levels were remarkably down-regulated in both rpfF_BC and cepI mutant strains,and the expression levels were restored with either addition of 20μM BDSF or AHL（C8-HSL）.The above experimental results showed that NlfR is a downstream component of the QS system regulatory network.Through electrophoretic mobility shift assay（EMSA）,we showed that AHL signal receptor protein CepR directly binds to the nlfR promoter and directly controls the expression of nlfR at transcription level.More interesting,NlfR regulates the expression of a series of overlapping genes co-regulated by both BDSF and AHL QS systems in B.cenocepacia.Although the BDSF system controls the expression of nlfR,the results of electrophoretic mobility shift assay showed that GtrR does not directly bind to the promoter sequence of nlfR.How the BDSF QS system regulates the expression of nlfR needs to be further explored.One possibility is that the BDSF system can indirectly regulate nlfR by controlling the AHL QS system;or there are other regulatory pathways,which need to be further investigated.The functional study of NlfR deepens our understanding of the QS system regulation network of B.cenocepacia.