Screening Of Plesiomonas Shigelloides Phage And Study On Activity Of Its Lysis System

Plesiomonas shigelloides is a common pathogenic bacteria in fish farming,which has strong pathogenicity to fish.At present,the problem of bacterial drug resistance is becoming more and more serious,so it is urgent to find a safe and original antibacterials that can replace antibiotics.Phage,as a bacterial virus,is specific and can quickly lyse host without affecting others.It is paid more and more attention as a new type of antimicrobial agents to control bacterial diseases of fish.In this study,a strain of virulent phage is isolated from the intestine of grass carp,named PSP01,using the Plesiomonas shigelloides as the host bacteria.PSP01 belongs to the Siphoviridae family,which is composed of an icosahedral symmetrical head and a long and noncontractile tail.The optimal multiplicity of infection of the phage PSP01 is 1.The one-step growth curve results show that the latent period of the phage is 30 min;the lysis period is 140 min;it enters a plateau from 170 min.PSP01 has good stability in the environment,showing good activity at p H 3-12,-20℃-50℃and 0.1%-3.5%Na Cl concentration for 1 h.Frist,after the whole genome of the phage PSP01 is sequenced and analyzed,it shows that the length of the genome is 97551 bp and the GC content is 41.65%.The genome contains a total of 152 open reading frames（ORF）,including 37 known proteins and 115 hypothetical proteins.These 37 known proteins act in structure,packaging,lysis,DNA replication and regulation and other functions.A phylogenetic tree is constructed based on the sequence of the terminase large subunit,showing that the phage is closely related to the Vibrio phage Ceto.Next,the activity of the lysis system of the phage PSP01 is studied,and it is found that the lysis cassette of the phage contains 3 genes,which code holin（HolPSP）,endolysin-1（Lys PSP-1）and endolysin-2（Lys PSP-2）.The three genes are located at ORF44（693 bp）,ORF45（675bp）and ORF53（441 bp）,respectively.HolPSP has no signal peptide（SP）and contains a transmembrane domain（TMD）;Lys PSP-1 has no SP and TMD;Lys PSP-2 has an SP at the N-terminal and no TMD.These three genes are separately expressed in Escherichia coli（E.coli）BL21（DE3）.HolPSP and Lys PSP-2 can reduce the OD₆₀₀value of E.coli,some red fluorescence appears via the cell viability measurement,and the color of the supernatant changes from colorless to yellow via theβ-galactosidase activity test,indicating that HolPSP and Lys PSP-2 have a lytic effect on E.coli.Lys PSP-1 does not reduce the OD₆₀₀value of E.coli during the expression process,the cell viability measurement shows green fluorescence,and theβ-galactosidase activity test shows no change in the color of the supernatant,indicating that Lys PSP-1 has no lysis effect on E.coli.HolPSP and Lys PSP-1 are co-expressed in E.coli BL21（DE3）,and it is found that the decrease in OD₆₀₀value of E.coli is more significant than that of HolPSP alone.Most of the red fluorescence appears,and the color of the supernatant is darker than that of HolPSP alone,which indicates that HolPSP and Lys PSP-1 have a synergistic effect on lysing E.coli.The HolPSP lacking TMD and Lys PSP-1 are co-expressed in E.coli BL21（DE3）,and it is found that the OD₆₀₀value of E.coli does not decrease,and the color of the supernatant has no change,indicating that TMD is also necessary for the lytic activity of HolPSP.Lys PSP-2 lacking SP is expressed in E.coli BL21（DE3）,the OD₆₀₀value of E.coli does not decrease,the color of the supernatant does not change,indicating that SP is also necessary for the lytic activity of Lys PSP-2.Finally,the protective effect of phage PSP01 on grass carp infection with Plesiomonas shigelloides is studied.Through intraperitoneal injection with pathogens and phages,the results show that the survival rate of grass carp injected with phage increases by 33.3%compared with grass carp without injection of phage.Thirty-six hours after injection,the grass carp spleen is taken for pathological observation,and it is found that the pathological symptoms are alleviated after injection with the phage.In summary,phage PSP01 is screened and its lysis cassette is studied.Meanwhile,it is evaluated the protective effect of phage PSP01 on grass carp infection with Plesiomonas shigelloides,which provides new data and theoretical basis for the use of phages to control fish pathogenic bacteria.