Study On The Regulation Mechanism Of An AraC-type Transcriptional Regulatory Protein In Burkholderia Cenocepacia

Quorum sensing（QS）is widely employed by bacterial pathogens to coordinate bacterial group behavior and regulate biological functions such as biofilm formation,motility,virulence.Burkholderia cenocepacia,a Gram-negative opportunistic pathogen,can cause life-threatening infections in susceptible individuals,particularly in patients suffering from cystic fibrosis.Quorum sensing in Burkholderia cenocepacia H111 mostly involves two signalling systems that depend on different signal molecules,namely N-acyl homoserine lactones（AHLs）and the diffusible signal factor cis-2-dodecenoic acid（BDSF）.Previous studies have shown that AHLs and BDSF control some similar phenotypic traits in B.cenocepacia.At present,there are many studies on the regulatory mechanisms of QS systems,but some recent evidences indicate that QS systems are also regulated by other regulatory factors,such as two-component systems,Ara C-type transcriptional regulators and other factors.Among them,the Ara C superfamily transcriptional regulators have been shown to regulate virulence and stress response in a variety of bacteria,for example,the similar regulatory mechanism to control QS signaling was confirmed in Pseudomonas aeruginosa.We screened and identified an Ara C-type transcriptional regulator,which was named as Atr A（Arac-type transcription regulate AHL）.An in-frame deletion mutant of atr A that we constructed was reduced in biofilm formation,motility and virulence.Complementation ofΔatr A mutant by the in trans expression of atr A restored biofilm formation,swarming motility and virulence to the levels of wild-type strain.Subsequently,we found that deletion of atr A also resulted in decrease in the expression level of cep I（AHL synthase encoding gene）at various growth stages,while increase of rpf F_Bc（BDSF synthase encoding gene）and bcl ACB（the gene cluster regulated by the QS）,which was confirmed by q RT-PCR analysis and RNA-seq analysis.Next,we measured QS signal production in the wild-type H111 strain,in-frame deletion mutant ofΔatr A,and the complementary strain.Our results showed that there was an obvious reduction in AHL signal production,but an increase in the BDSF signal production in theΔatr A mutant compared with that in the wild-type strain.To test if transcriptional regulation of target genes is achieved by direct binding of Atr A to their promoters,electrophoretic mobility shift analyses（EMSA）were performed.We found that Atr A regulates target gene expression by directly binding to promoters.The exact binding site of cep I promoter was also determined by using the DNase I footprinting methods as（GCG CGC TGT AAT GCA CGC ATA CAA AAG CAC AGA TCCG）.We continued to analyze and compare the transcriptomes of the wild-type strain,theΔatr A mutant strain andΔcep I mutant strain by using RNA-Seq.We compared the transcriptome profiles of theΔatr A mutant and theΔcep I mutant and found an overlap in their regulated genes.Moreover,the atr A and cep I mutants also showed similar biological phenotypic defects,including a significant decrease in biofilm formation,motility and virulence and QS signal production.At the same time,we also found that the gene bcam0582,in the vicinity of atr A,can control the expression level of bcl ACB.Deletion of bcam0582 resulted a significant increase in the expression level of bcl ACB.In summary,the Ara C superfamily transcriptional regulator Atr A can participate in the regulation of the QS systems of B.cenocepacia and directly affect the synthesis of QS signals.The gene bcam0582 can also affect the expression level of bcl ACB gene cluster.However,the regulatory mechanism is unclear,and the further exploration is needed.