Identification And Characterization Of The Virulence Regulatory Mechanisms In Dickeya Zeae EC1

The rice foot rot is an important disease caused by Dickeya zeae,which is one of the most threating bacterial diseases.D.zeae EC1 is one of Dickeya isolated that can infect both monocotyledons and dicotyledons,whereas most other Dickeya species and isolates can only infect dicotyledons.The phytotoxins zeamine and zeamine II are considered to be an important determinant in hosts range and pathogenesis.In addition,zeamines have a potent antimicrobial activity against a range of gram-negative and gram-positive bacteria and fungal pathogens.Previous studies showed that the zeamine synthetic gene cluster is composed of 18 coding genes,among them,zmsA is essential for synthesis of both zeamine and zeamine II,and zmsK is necessary for synthesis of zeamineII.However,the potential signaling pathway and regulatory mechanisms that govern the biosynthesis of zeamines remain largely unknown.In this research,we set to identify the potential transcription factors and putative quorum sensing systems that influence zeamine production through deletion analysis of target genes.The preliminary screening results from the mutants of 18 genes encoding 7 families transtripional regulatory factors indicated that the mutation of fis decreased zeamine production by about about 70% compared with the wild-type strain EC1.The product of fis belongs to the widely conserved Fis family transcriptional regulators.Similarly,production of extracellular enzymes and biofilm formation are reduced significantly in the fis mutant.In addition,we found that the mutants of ohrR and emrR were decreased in zeamines production,biofilm formation and cellulase activity.Both OhrR and EmrR belong to the MarR family transcriptional regulators.There results suggest that Fis,OhrR and EmrR are involved in regulation of zeamine production and other virulence factors in D.zeae EC1.Complementation analysis confirmed that Fis positively regulated the production of zeamines.Transcriopome and phenotype analysis showed that Fis was a potent global transcriptional regulator modulating various virulence traits,including production of extracellular enzymes and exopolysaccharides,biofilm formation and cell aggregation,swaimming and swarming.DNA gel retardation analysis showed that Fis directly modulated the transcription of quorum sensing regulator gene vfmE and key virulence genes through DNA/protein interaction.Meanwhile,it was also found that the transcriptional expression of fis was modulated by SlyA,suggesting its location at the down-stream of SlyA in the virulence regulatory networks.Consistent with the regulatory role of Fis on virulence gene expression,bioassy showed that the fis mutant was markedly attenuated in the virulence against rice seed germination.Similarly,complementation analysis confirmed that OhrR also played an important role in regulation of zeamine production.The results of qRT-PCR showed that expression of zeamine biosynthesis genes zmsA and zmsK are reduced markedly in the ohrR mutant,compared with its wild type strain.In addition,OhrR also regulated the cellulase production and cell motility.To understand the impact of these characterized regulators on bacterial virulence,double and triple deletion mutants were generated.The results showed that the double deletion mutants of ?(ohrR+slyA)and ?(ohrR+fis)produced less zeamines than the corresponding mutants ?ohrR and ?slyA,and the triple deletion mutant ?(ohrR+slyA+ fis)produced lesser zeamines than the corresponding single and double mutants.The results of EMSA showed that OhrR could directly bind to the protomer region of slyA and fis to regulate the transcription of slyA and fis.Bioinformatic analysis showed that D.zeae strain EC1 contains a gene cluster homologous,which the vfm quorum sensing gene cluster identified in D.dadantii 3937.These vfm genes were deleted individually in strain EC1,and the results showed that production of zeamines and extracellular enzymes was decreased significantly in most of these vfm genes.The zeamine production deficient phenotype of ?vfmA??vfmW and ?vfmK could be restored by the cell-free supernatants from the wild type strain EC1.However,the vfm signals from D.dadantii 3937 could not be detected using the reporter strain of D.zeae EC1,suggesting that the vfm signal of EC1 might be different from the vfm signal of 3937 strain.The results of qRT-PCR showed that the expression of vfmE?vfmA?vfmK?zmsA and zmsK was decreased markedly in ?vfmH,and the expression of vfmH?vfmA?vfmK?zmsA and zmsK was decreased markedly in ?vfmE.Time curve analysis showed that vfm signal level was increased along with the bacterial proliferation,but decreased sharply when the signal reached a threshold value,suggesting that strain EC1 may produce a Vfm degradation or modification enzyme.Preliminary results showed that the vfm quorum sensing signal was highly polar,weak thermal stability in water,and stable in various pH solutions.The data of column chromatography and HPLC analysis showed that strain might produce more than one vfm signal anologues.This study revealed that Fis global transcriptional regulator modulated production of multiple virulence factors in D.zeae strain EC1.In addition,two MarR families transcriptional regulators alsoplayed a role for regulation of the virulence in strain EC1.Furthermore,a new vfm-like quorum sensing signal was found in strain EC1,which could be structurally different from the vfm quorum sensing signal of 3937.