Function Of Genes Cys B,cys J And SEN1393 In The Recovery Of Sublethal Salmonella Enteritidis Cells

Salmonella enterica serovar Enteritidis（S.Enteritidis）is the major pathogens causing foodborne diseases,and eggs are the main carrier of infecting human.Although the egg white contains antibacterial substances such as lysozyme,conalbumin,and protease inhibitors,S.Enteritidis could still thrive in it.Therefore,researching the survival mechanism of S.Enteritidis in egg white could provide an effective strategy to solve the food safety problems caused by it.At present,the stress response-related thioesteras gene ybg C,the inner membrane protein gene yoa E,and the heat shock protein gene htr A of S.Enteritidis have been studied,but the gene related to the sulfate assimilation pathway have not been reported.Previous studies have shown that specific gene SEN1393 only exists in S.Enteritidis,and the deletion of it could affect the metabolic ability of S.Enteritidis,and also resulted in the down-regulation of gene cysJ expression.Gene cysJ is involved in the SO₃^2-reduction in the sulfate assimilation pathway,and affected by the sulfur metabolism regulation gene cysB.Hence,ΔcysB andΔcysJ were construction via homologous recombination of S.Enteritidis standard strain ATCC13076.The survival and recovery characteristics in egg white ofΔcysB,ΔcysJ,ΔSEN1393 and wild-type S.Enteritidis were explored,the growth of strains in different compounds environments were analyzed.Results could provide experimental evidence to elucidate the molecular mechanism of S.Enteritidis survival in egg white.The main researching contents and results are as follows:（1）Construction of in-frame deletion mutantsΔcysB andΔcysJ.Primers with the restriction enzyme cutting site were designed according to the upstream and downstream sequences of genes cysB and cysJ,respectively,and the whole homologous arm was amplified by PCR.The homologous arms of cysB and cysJ were 839 bpand 757 bp,respectively Homologous recombinant intermediate plasmids pMD₁₉ΔcysB and pMD₁₉ΔcysJ were constructed using pMD19-T Vector.Then SacⅠand XbaⅠrestriction enzymes were used to digest the plasmid and pRE112 vector,respectively.After treated by T4 DNA Ligase,the plasmid was transferred into E.coli SM10λpir competent cells to obtain homologous recombinant plasmids pREΔcysB and pREΔcysJ.Subsequently,the plasmid was transferred into S.Enteritidis competent cells by electroporation（2500 V,5ms）,respectively,and thenΔcysB andΔcysJ were obtained by sucrose selection.（2）Function of genes cysB,cysJ and SEN1393 in S.Enteritidis recovery.ΔcysB,ΔcysJ,ΔSEN1393 and wild-type S.Enteritidis were treated with 25.51 mg/m³ ozone for 30 min,and the viability,the cell activity,the recovery ability in egg white environment and changes of sulfides were observed.The results showed that:a)Under the normal culture conditions,the number of mutant and wild-type strains grew upto 8 log CFU/m L.After ozone treatment,the number of four strains decreased,andΔcysJ decreased to 4.7 log CFU/m L,which was significantly higher than that of wild-type strains.b)The activity of the strain decreased after ozone treatment,ΔcysJ was the most damaged.（c）The number ofΔcysB decreased by 6.4 log CFU/m L when the normal strain was transferred to egg white,which was significantly higher than other strains.Ozone-treated sublethal cells were cultured in egg white for 24 h and recovered in large numbers.The growth ofΔcysJ andΔSEN1393 was larger than wild type,1.9 log CFU/m L and 1.3 log CFU/m L,respectively,while the growth ofΔcysB was significantly lower than wild type.d)The deletion of genes cysB and cysJ significantly reduced the Thioredoxin Reductase（TrxR）activity and endogenous H₂S content.Although the TrxR activity and H₂S content increased after oxidative stress stimulation,they were always significantly lower than those of the wild type.The TrxR activity and H₂S content ofΔSEN1393 were also lower than those of wild type after 24 h incubation in egg white.（3）Screening of compounds promoting strains recovery.The ozone-treated sublethal strains were transferred to M9 medium,and different inorganic salts,amino acids and egg white proteins were added to the medium,respectively,to observe the recovery of the strain.The results showed:a)After added 5 g/L Na₂Se O₃ and Na₂S₂O₃,the OD₆₀₀ values ofΔcysB andΔcysJ were similar to those of the wild type,about 0.8.b)After 0.15 mg/m L supplementation of cysteine,methionine,alanine,histidine,arginine and glutathione,the growth rate of mutants were higher than wild type.c)ΔcysB,ΔcysJ andΔSEN1393 were enhanced by conalbumin and apoovotransferrin,and OD₆₀₀ values ranged from 0.71 to 1.00after 24 h culture.In conclusion,genes cysB,cysJ and SEN1393 are related to the growth of S.Enteritidis in egg white,and their deletion significantly influences the recovery ability of S.Enteritidis.Addition of Na₂S₂O₃,cysteine,methionine,conalbumin and other compunds could improve the recovery ability of mutants.