Genes Expression Regulation By PhoP/PhoR Two Component System And The Integrases Functions Of Genomic Islands In Riemerella Anatipestifer

Infectious serositis of duck is an acute,contagious and septic infectious disease caused by Riemerella anatipestifer?R.anatipestifer,RA?,which occurs primarily in 1 to8-week-old ducks but is most common in more susceptible 2 to 3-week-old ducklings.The disease is characterized by pericarditis,perihepatitis,airsacculitis and meningitis.It is one of the most serious bacterial infectious diseases endangering duck industry.At least21 RA serotypes have been reported,and no effective cross protection between serotypes.Because of high mortality,growth retardation,quality decline,low feed return rate,then increased treatment costs and the duck industry had been caused huge economic losses.Based on the analysis of bioinformatics in the early stage of our research,it is predicted that RAYM?RS09735 and RAYM?RS09740 are the typical two component system?TCS?of R.anatipestifer.A double genes mutation strain YM?RS09735/RS09740 had been constructed.Compared with the wild strain,YM?RS09735/RS09740 mutant strain is significantly reduction the virulence to the duckling.However,the regulatory mechanism on the virulence of R.anatipestifer TCS is still unclear.This research main contents and results are as follows:1. Transcriptome analysis of RAYM?RS09735/RAYM?RS09740 genes mutation strain and the effect on gene expression of R.anatipestifer RA-YMRNA?Seq showed that 112 genes were up regulated and 693 genes were down regulated.Ten differentially expressed genes were randomly selected and the transcriptome sequencing results were verified by q PCR.The data showed that q PCR results were consistent with the transcriptome sequencing results,which confirmed the accuracy of RNA?Seq data.Go enrichment analysis showed that the differentially expressed genes involved biological processes,cell components and molecular functions.KEGG pathway analysis showed that RAYM?RS09740 was PhoP protein,indicating that RAYM?RS09735 and RAYM?RS09740 constituted PhoP/PhoR two-component system.PhoP/PhoR is a novel two-component system first reported in gram negative bacteria.Transcriptome data showed that the expression of peptidoglycan hydrolase Nlp/P60gene was down regulated significantly.Gel shift assay showed that pured PhoP protein could bind to Nlp/P60 gene promoter in vitro.The results suggested that PhoP protein can directly regulate the expression of Nlp/P60 gene.Nlp/P60 gene mutation strain was constructed,and the virulence of ducklings was 1.33 times lower than RA-YM strain.Nlp/P60 gene is a virulence related gene of R.anatipestifer.2. Construction of phoP and phoR gene deletion strains and their effects on gene expression and regulation of R.anatipestifer RA-YM?phoP and?phoR genes deletion mutants were constructed by conjugation transfer.The results showed that there was no significant difference in the growth rate of the mutant strains and RA-YM wild strain in liquid medium.The results of animal experiments showed that the LD₅₀ of wild strain RA-YM,?phoP and?phoR were 3.98×10⁴CFU,1.88×10⁹CFU and 4.22×10⁹CFU,respectively.The virulence of the mutant strains of?phoP was about 4.7×10⁴ times lower than the wild strain,and the virulence of the mutant strain of?phoR was about 1.0×10⁵ times lower than the wild strain.The results showed that the tissue bacterial load of?phoP,?phoR gene mutant strains were significantly lower than RA-YM wild strain in 24 h and 48 h.According to the histopathological observation of the Ducklings infected with the gene mutation strains for24 hours and 48 hours,no obvious pathological changes were found,which confirmed that both the genes of phoP and phoR were closely related to the virulence of RA-YM strain.Through the comparative analysis of the transcriptome differentially expressed genes of RA-YM strain,and?phoR mutation strains,the results showed that 186 differentially expressed genes comparative with RA-YM strain and?phoP,of which 126 genes were up regulated and 60 genes were down regulated in?phoP.There are 243 differentially expressed genes comparative with RA-YM strain and?phoR,of which 97 are up regulated genes and 146 genes are down regulated.3. RAP44 phage integrase mediated 50K genomic island integration of R.anatipestiferThrough the analysis of bioinformatics of transcriptome,78 consecutive genes were found to be differentially expressed,and the 78 consecutive genes were located in the50Kb gene cluster area.This gene cluster with 19bp direct repeats at both ends,and located in the downstream of t RNA Ser.The gene cluster was named 50K genomic island.Through the whole genome sequence alignment,it was found that part or complete genomic island were found in R.anatipestifer RA-YM,HXb,Yb2,RA-GD,ATCC 11845,CH-3,CH-1 and other virulent strains.The genomic island was derived from R.anatipestifer virulent phage RAP44.We found that this island could form intermediate cyclization molecule.The integrase structure was similar to Cre enzyme.Using the integration characteristics of integrase.We constructed a recombinant suicide integration plasmid p RE112-spec-int,which was introduced into R.anatipestifer by conjugation and transfer,and it was found that the integrase mediated precise site-specific integration.4. Identification of an integrase that responsible for precise integration and excision of R.anatipestifer 10K Genomic IslandThrough bioinformatics analysis of R.anatipestifer ATCC 11845,we found that there was another 10K genomic island besides 50K genomic island,but RA-YM strain did not contain this genomic island.Based on the integration characteristics of integrase,we constructed a recombinant suicide integration plasmid,which was introduced into R.anatipestifer RA-YM strain by conjugation and transfer,and a stable integration strain was obtained.PCR detection showed that the integrase mediated precise integration and excision.Furthermore,the e GFP expression plasmid was constructed based on the integration characteristics of the integrase,and the recombinant RA-YM strain expressing e GFP was obtained by conjugation and transfer.In summary,this study is the first to elucidate the role of PhoP/PhoR TCS in R.anatipestifer gene expression regulation,providing a new perspective for further study on the role of TCS in the regulation of virulence of R.anatipestifer.A stable genetic operating system was studied and established by using integrase genomic island,which provided a new method for the expression of foreign genes and the construction of complementary strains,and laid a foundation for the horizontal transfer and evolution of the genome.