Construction of an rpoS knockout mutant of Salmonella enteritidis and characteristics of its growth

Objectives To construct a mutant by knocking out the rpoS gene of Salmonella enteritidis with the help of a λ-red recombinant system,to investigate the growth characteristics of the rpoS knockout mutation in S.enteritidis under stress conditions,and to discuss the biological effects of the rpoS gene.Methods A kanamycin-resistant gene flanked by homologues of the rpoS gene was amplified with PCR and electroporated into S.enteritidis carrying the plasmid pkD46.After the rpoS gene was replaced with the kanamycin-resistant gene,S.enteritidis/rpoS strains were screened and then amplified with PCR.Results An rpoS knockout mutant of S.enteritidis was successfully constructed.There were no significant differences(P0.05)in the growth curves of the rpoS mutant and wild-type strains.S.enteritidis/ rpoS strains had significantly less ability to survive(P0.05)compared to wild-type strains under stress conditions of 45 ℃,2% NaCl,and pH 4.0.Conclusion An rpoS knockout mutant of S.enteritidis was successfully constructed.Expression of the rpoS gene was induced.The rpoS gene plays an important role in the ability of S.enteritidis to overcome stress in the form of high temperatures,high osmolarity,and an acidic environment.This study provided basic data for further study of the mechanism of action of the rpoS gene in response to stress.