Studies On The Regulative Mechanism Of Repressor Protein LexA Involved In The Virulence Of Pseudomonas Plecoglossicida

Large yellow croaker（Larimichthys crocea）is one of the four major marine products in China,which has high economic value.Intensive cultivation of large yellow croaker has brought serious diseases."Visceral white spot disease"is one of the most common diseases in cage cultured L.crocea in recent years.After infection,white nodules appear in the viscera of L.crocea,and the mortality of infected L.crocea is very high,which has caused serious economic losses.At present,Pseudomonas plecoglossicida is widely recognized as the pathogen of visceral white spot disease in L.crocea.P.plecoglossicida is a typical opportunistic pathogen widely distributed in seawater.P.plecoglossicida can not only infect L.crocea,but also infect many other aquatic economic fishes.In recent years,the incidence rate of P.plecoglossicida infection has increased significantly,which has brought huge economic losses to China’s aquaculture industry.In our previous study,P.plecoglossicida NZBD9 was isolated and identified from the spleen of L.crocea with"visceral white spot disease".It was found that lexA might participate in the virulence regulation mechanism of P.plecoglossicida with dual RNA-seq analysis.LexA was firstly identified as a key transcription factor in SOS response in E.coli,involved in DNA repair,and lexA gene exists in almost all bacteria.However,studies on the involvement of LexA in the regulation of bacterial virulence are still rare.Therefore,this study is a preliminary study on the role and molecular mechanism of lexA gene in virulence regulation of P.plecoglossicida.The main contents of this study are as following:（1）Temperature,pH,mitomycin-C,H₂O₂ and heavy metals were used to treat P.plecoglossicida NZBD9,and the expression of lexA gene was detected by q RT-PCR.The results showed that the expression level of lexA was significantly down regulated at 4℃and up-regulated at 44℃;the expression level of lexA was remarkably down regulated at pH4 or 9;the expression level of lexA was down regulated under different concentrations of H₂O₂;the expression level of lexA was notably up-regulated under mitomycin-C;the expression level of lexA was significantly down regulated under Cu²⁺and Pb²⁺stression.These results indicate that these stress treatments have a significant effect on the expression of LexA at the transcriptional level,indicating that LexA plays an important role in the response to various environmental stresses other than DNA damage inducers.It is worth noting that in this study,as a DNA damage inducer,mitomycin C promotes the transcription and expression of LexA,which may be a feedback regulation mechanism after LexA participates in DNA damage repair through self cleavage and degradation.（2）By using homologous recombination technology,we constructed the lexA mutant strain,and detected the adhesion,biofilm formation,movement,hemolysis and growth ability of the mutant strain,and analyzed the effect of lexA mutation on virulence phenotypes of P.plecoglossicida.The results showed that the adhesion ability and biofilm formation ability of△lexA strain were significantly decreased,while the motility,hemolysis and growth ability of△lexA strain were not significantly different from those of the wildtype strain.These results indicate that the absence of lexA can obviously reduce the adhesion and biofilm forming ability of P.plecoglossicida,indicating that lexA is involved in the virulence regulation of P.plecoglossicida adhesion and biofilm formation.（3）In order to further understand the regulatory mechanism of lexA on the virulence of P.plecoglossicida,we performed RNA-seq on△lexA.Meanwhile,we carried out chromatin immunoprecipitation sequencing（Chi P-seq）around LexA,and analyzed the correlation between the results of Chi P-seq and RNA-seq,and obtained potential target genes including lrg A,ton B,and the potential target genes were verified by q RT-PCR.The potential LexA-interaction proteins including Rec A,Htp G and Fur were identified by Co IP-Ms.These results suggest that LexA may affect the virulence of P.plecoglossicida by regulating the expression of downstream genes,together with Rec A,Htp G and Fur.