Studying On Related Gene Deletion Of HPI In E.Coli Strains

High pathogenicity island of Yersinia (high pathogenicity island, HPI) was first found in Yersinia which size from36kb to43kb, encoding the gene of uptaking siderophore Yersinia bacterin (yersiniabactin. Ybt) and its regulation gene(irp1, irp2, fyuA). HPI is related to iron uptake and regulation, and is the essential genetic unit of expressing mouse lethal phenotype; It is one of the important factors to determine the strain pathogenicity and virulence. Actually to say, HPI is presented in a variety of intestinal bacteria, such as E.coli, Salmonella, Klebsiella, HPI is distributed in different species by level transferring of genes. HPI not only gives a special virulence in the pathogen relating the infection process, but also plays an important role in the process of bacterial evolution. To reveal the virulence of HPI pathogenicity island of E.coli, this passage makes three aspects of research:First, knock back single gene in four genes of two HPI bacterias with the technique of the Red recombination system, screen out the knock back strains successfully. The four key structural genes irp1, irp2, fyuA, ybtA belong to HPI pathogenicity island which are closely related to the toxicity of the pathogenicity island, this experiment has firstly synthesized part of the PCR products of the four genes which containing the recombinant homologous arm. using Red recombination system, we expression induced protein under30℃, recombine the PCR products with single-gene deletion mutant to exchange genes. Screen out the strains of successful reorganization, the successful reorganization of bacteria don’t have any selection marker, and unsuccessful reorganization of bacteria have the FRT fragment marked the Kanamycin resistance, so the experiment use the large number of spotting to screen out successful reorganization strains step-by-step.Compare the growth trend of four single-gene deletion mutants (△irp1,△irp2,△yhtA,△fyuA) of S793103with the original strain in iron-deficient NBD medium to study its iron uptake changes. To study ferritin regulatory function of the swine HPI+E.coli pathogenicity island key structural gene further, we use iron-deficient culture medium NBD to cultivate original wild strains and gene deletion strains, and to study their growth characteristics by viable counts, we use NBD medium to continue monitoring10hours till reach the stability of bacterial growth, and during the measurement we observe the number of OD600, map out a growth trend graph to compare their iron uptake function of knock-out and knock-back. The results show that there is a certain influence in growth rate, which indicates that the HPI pathogenicity island is related to the iron uptake ability of the bacteria, and the time reach the growth plateau of5bacterias have no significant differences, suggesting that the HPI+E.coli may also have the other iron uptake system. Third, we study virulence of the single-gene deletion strains and wild strains of E.coli by animal experiments, which verifies the role of four key genes related to E.coli, and it settles substantial fundament for further research HPI+E.coli derived from pigs. Grouping the30-day-old ICR mice, inoculate mice with original wild strains and deletion mutants of the four genes in different concentration gradients, and set the positive and negative control, observe every12hours after inoculating for7days to compare virulence changes of the gene which knock out or back by calculating the LD50of mice. Autopsy, record the naked eye pathological changes in the death of mice, bacterias are isolated and virulence genes are identified by PCR. Data analysis show that the virulence to mice of original wild strains and gene deletion mutants mice is significantly different, pili strains’ pathogenicity is significantly stronger than strains without fimbriae, which suggest that HPI pathogenicity island is related to the virulence of E.coli.