The Characteristics Of Avian Pathogenic Coliphage And The Selection Of Genes Related With Phage Receptor

APEC is an extraintestinal pathogenic Escherichia.coli which can cause local or systemic infection of chicken,turkey and other avian species after infection.The pathogen strains,which led to huge economic losses to the poultry industry around the world,mainly caused respiratory infections,polyserositis,pericarditis,airsacculitis,septicemia and other serious parenteral infections.75 avian coliphages were isolated in the experiment and we did a series of biological characteristics analysis using part of coliphages.Experimental researches such as genome sequencing,comparative genomic analysis and the preliminary exploration of replacement of tail fibers were finished by using temperate phage P88.We selectd one APEC strain named XM and T4-like phage QL01 to screen genes associated with phage receptors by using Tn5 transposon insertion deletion mutagenesis.Three nullmutant strains were constructed by using the recombineering method of scarless mutagenesis.The experiment provided a new idea for exploring the receptors of T4-like phage and the mechanism of the phage infection with E.coli.1.Isolation,Purification and Biological Characteristics analysis of Phages from Escherichia coliThis experiment obtained 75 avian pathogenic E.coli phages from duck feces samples in a certain vegetable market of Nanjing by using double-layer agar plate method.By measuring the host spectrum of these phages,it was found that different phages can lyse 2-33 strains.We did a series of biological characteristics analysis such as the heat stability,the MOI and other tests using part of phages.And we observed that the diameter of some phages can reach to 5mm compared the adaptation of 11 phage strains under different temperature and point-in-time conditions.Three phage strains were observed by transmission electron microscopy.The results showed three phage strains belongs to T4-like phage viruses genus,Myoviridae family.2.The isolation of bacteriophage P88 and the genome analysisTwo bacteriophages,named P88 and pro147,were obtained following mitomycin C induction of 54 lysogenic strains by using double-layer agar plate method,they all belong to P2-like phages.The genome of phage P88 was sequenced by Illumina MiSeq.The result revealed that the total length of P88 genome consists of a double-stranded 35814bp with the average GC content of 52.9%,and 53 putative ORFs were predicted without tRNA.Comparative genome analysis of P88 and pro 147 showed that they have significant difference with phage P2,covering 5%,ident 90%and covering 75%,ident 97%,respectively.And the P88 genome exhibited low similarity with pro 147,covering 6%,ident 92%.However,the tail fiber protein sequence of P88 shares high similarity with that of pro 147,covering 100%,ident 70.Alignment of the P88 tail fiber protein with that of pro147 shows that there were two fragments of hypervariable regions of 120 aa(576-695 in P88)and 31 aa(716-746 in P88)in the C-terminal region.we speculated that the hypervariable regions may have an impact on the host spectrum of two phages,and the comparative genome analysis of tail fiber protein of P88 and pro 147 provide theoretical foundation for the following experiments.3.Tn5 transposon screening genes associated with phage receptorsTn5 transposon insertion deletion mutagenesis is an important gene manipulation tool technology in genome analysis.Transposon inserted into genome can possibly lead to gene inactivated at insertion site.The experiment utilized this characteristic of Tn5 transposon to establish a mutant library and screen genes associated with avian pathogenic Escherichia coli T4-like phage receptors.The Escherichia coli S17-1(Xpir),which carries the PUT-Mini-Tn5-Km plasmid,was used as donor strain,and the Escherichia coli XM,which induced by nalidixic acid,was used as the recipient strain.The successful strains were screened from cultivated strains by using double-layer agar plate method(ampicillin resistance and nalidixic acid resistance).Three mutants,which may be related to phage receptors,were screened by using phage QL01.The reverse primers were designed by Tn5 transposon,and the relative gene sequences were analyzed.The result revealed that two mutants were associated with lipopolysaccharide(LPS)and peptidoglycan(PG).Three nullmutant strains were successfully constructed by using the recombineering method of scarless mutagenesis.When using phage QL01 to infect different strains,we can see obvious lysis region in the plate growing with wild strain XM,and we can not see any plague in the plate growing with mutant strains that inserted with Tn5 transposon while the nullmutant strains can also be lysed by phage QL01,but the plaques were blurry.This lytic phenomenon of three types of strains can be steadily observed by repetitive experiment tests.