Construction Of Escherichia Coli O157:H7 Gene Deletion Strains And Its Biological Characteristics

When exposed to adverse environment,Escherichia coli(E.coli)O157:H7 enters the viable but nonculturable(VBNC)state,resulting in the omission of foodborne pathogen during detection.Therefore,here we constructed three mutants of E.coli O157:H7 regarding gene fimC,luxS and ropS,and analysed the differences between the wild-type and the mutants in their VBNC state.The research contains three parts: 1.Construction of the E.coli O157:H7 gene-deficient strains by using the lambda RED recombination technology;2.Preparation of VBNC state cells of E.coli O157:H7 as well as its mutants,by combining low temperature and eutrophication,and 3.Phenotypic and physiological characterization of E.coli O157:H7 mutants.The gene knockout protocol of the subject: Genes fimC,luxS and rpoS,involved in VBNC state formation,were disrupted by RED recombination as follows: 1.PCR amplification of the kanamycin resistance gene with upstream and downstream homologous regions of the targeted genes;2.The PCR fragments were electroporated into E.coli O157:H7,which harbors plasmid pKD46;3.Induction of the expression of the Red recombinase to integrate the foreign DNA fragment into the target genes;4.Induction of the expression of the FLP recombinase to excise the kanamycin resistance genes that were flanked by FRT sites.Finally,the three target genes were successfully deleted,and here designated as E.coli O157-?luxS,E.coli O157-?rpoS and E.coli O157-?fimC were obtained.On the other hand,we compared the differences between the wild-type E.coli O157:H7 and gene-deficient strains in VBNC state.During the VBNC induction process,the cells were monitored by AODC,DVC and plate counting.The data showed that: E.coli O157-?rpoS completely entered VBNC state after-20 °C eutrophication induction on the 18 th day,which was half of that for the wild-type and other gene defective strains;The total number of bacteria was stable,and the number of viable cells was 1 to 2 magnitudes lower than the total number of bacteria;The shape of the bacteria changed from rod to sphere.Finally,the differences of biological characteristics between wild-type E.coli O157:H7 strain and gene deletion strains were studied.It was found that there were no genetic differences in the gene-deficient strains that were passaged 10 times,compared with the first-generation gene deletion strains;the loss of the fimC gene could cause a decrease in the motility of the E.coli O157:H7 strain;the deletion of rpoS gene severely reduced the ability of E.coli O157: H7 to cope with acid stress;and compared to wild-type E.coli O157:H7,the deletion of the fimC and luxS gene affected the formation of bacterial biofilms to some extent.Our study aimed at the mechanisms of VBNC state formation regarding foodborne pathogen E.coli O157:H7,analysed the roles of gene fimC,luxS and ropS,on the molecular level,and the results contribute to the safety of food processing technology,and to provide a reliable reference for the prevention and control of foodborne pathogens.