Function Research Of Transcriptional Regulator BfmR In Pseudomonas Aeruginosa

The transcriptional factor BfmR plays an important role in controlling the biofilm formation by regulating phdA which is involved in cell lysis and eDNA release,making a transition to maturation-1 stage.BfmR plays a positive role in regulating biofilm formation.In addition,BfmR inhibits rhl Quorum Sensing(QS)system,reduces the production of virulence factors(pyocyanin pigment,rhamnolipid),and attenuates bacterial acute infection.However,the global effect of BfmR as a transcriptional regulator remains unknownUsing chromatin immunoprecipitation(ChIP)followed by high-throughput sequencing(seq),we identified that BfmR binds to at least 174 loci on MPAO1 genome Combined with the results of RNA-seq experiments,we revealed that 172 genes are BfmR potential targets engaged in antibiotic resistance,biofilm formation,QS system and metal homeostasis.We verified the binding of BfmR to the six sites by electrophoretic mobility shift assays(EMSA).Moreover,the conserved binding site of BfmR was identified through MEME suite,was similar with conversed sequence(GATACAnnAC)reported in previous study,and demonstrated by DNase I Footprinting assays in vitroAttractively,BfmR binds to the intergenic regions of pa5359-phoB and czcC-czcR,and regulates the promoter activity of czcC,czcR and phoB,we confirmed that the binding site is essential for BfmR to activate phoB according to the deletion in binding site and subsequent Western blot assays.besides the relevant experiment proved that phoB is required for BfmR regulating biofilm formationFurthermore,we found that BfmR binds the promoter region of part of CzcR-and PhoB-regulated genes,and influence the expression of some of genes(e.g.,czcC,pqsA,vreA and rhlR).This suggests that BfmR may achieve a wider range of regulation through this pathway,forming its complex regulatory networkFinally,we identified 10 binding sites of BfmR in DK2 strain.There exsits homologous gene in MPAO1 strain for most genes,such as bfmR,oprL,and nalC.Three binding sites of BfmR,DNA fragments are the acquisition of genetic material for adaptation of bacterial pathogens to a permanently host-associated lifestyle for DK2,suggests that BfmR may play an important role in the evolution of Pseudomonas aeruginosa.Taken together,this study combined the techniques ChIP-seq and RNA-seq to define the potential BfmR regulon,we found that BfmR is involved in many important biological processes.The conclusion provides an important basis for deeply investigate the function of BfmR.Besides,we further explored that phoB is the essential component of biofilm formation regulated by BfmR,provided new insight of biofilm formation mechanism.