Locating And Charactering Replication Region Of Virulence Plasmid PEIB202 In Edwardsialla Piscicida

Aquatic animals infected by Edwardsialla piscicida EIB202 can lead to the development of edwardsiellosis,which caused enormous economic losses worldwide.We previously developed an attenuated live vaccine WED based on virulent strain EIB202.Turbots vaccinated with WED via intraperitoneal injection can effectively protect themselves from edwardsiellosis,while inoculation by immersion had an undesirable protective effect.To search for colonization-associated genes,we tested virulent-related phenotypes of EIB202,WED and ΔTTSS,ΔaroC,ΔpEIB202,ΔTTSS/ΔaroC,ΔTTSS/ΔpEIB202,andΔaroC/ΔpEIB202 mutants.Our results showed that pEIB202 played a key role in biosynthesis of LPS and colonization of E.piscicida on zebrafish.However,there are 53 hypothetical ORFs in pEIB202 which has more than one copy in E.piscicida.Thus it was difficult to identify colonization-associated ORFs.And it’s necessary to locate the replicon of pEIB202.Firstly,pEIB202 were digested by Hind Ⅲ and Pst Ⅰ and subclones pMHJE10 and pMPJE8 were obtained,which overlapped 24 661 bp(41 970-22 927 bp).Secondly,pEIB202 were digested by Hind Ⅲ/Pst Ⅰ and Hind ⅢNhe Ⅰ and then pAPH8 and pANH16 were obtained,which overlapped 16 520 bp(6 408-22 927 bp).At the same time,we got accurate location of replicon by Sau3A Ⅰ partial digestion,which located between 18 249 and 21335 bp(3 087 bp).Finally,PCR was performed and minimal subclone pRep-q77(18 837-20 816 bp,1980 bp)was identified,including ETAE RS16765(transcriptional repressor)and ETAE_RS 16770(replication protein).The replication protein was highly identical to that of Piscirickettsia salmonis,and there was primase and DNA-binding domain on it,which suggested that pEIB202 could unwind independently.Besides,the transcriptional repressor was identical to antidote protein of Nostoc punctiforme,implying the plasmid replication and maintaince function.We tested the copy numbers and stability in both E.piscicida and E.coli of the replicon and results showed that pEIB202 and subclones were low-copy in E.piscicida,which were still higher than those in E.coli.Also,copy numbers of pMHJE10 and pMPJE8 with higher molecular sizes were relatively low in E.piscicida and E.coli.And 49%E.piscicida and 25%E.coli carried the minimal plasmid pRep-q77 after 2 generations antibiotic-free culturing.Furthermore,we tested the contribution of pMHJE10 and pMPJE8 to the colonization on mucosal tissues of zebrafish.Results showed that the survival rates of zebrafish injected with pMHJE10/ΔpEIB202 and pMPJE8/ΔpEIB202 were similar with those injected with EIB202 and ΔpEIB202.Meanwhile,colonization of pMHJE10/ΔpEIB202 and pMPJE8/ΔpEIB202 on mucosal tissue of zebrafish by immersion were 100 times lower than that of ΔpEIB202,much lower than that of EIB202,suggesting that plasmid pEIB202 played an important role in mucosal colonization of E.piscicida.The location of replicon laid a foundation for investigation into the gene function of pEIB202.And the pilot study of pMHJE10 and pMPJE8 on virulence and colonization shed light on improving the protective effect of vaccine via immersion vaccination.