The Expression Characteristics Of Mig-14 In Salmonella Enterica Serovar Typhi

Objective:mig-14 is a horizontal-transfer acquired gene of salmonella,plays an important role in resistance of bacteria to polymyxin B.Furthermore,MIG-14,the expression product of mig-14,acts as a regulator to regulate a series of genes.However,despite the critical role of mig-14,the expression mechanisms and characteristics of mig-14 remain unknown.This study aims to investigate the expression mechanisms and characteristics of mig-14 in Salmonella enterica serovar Typhi in different environments.Methods:1.The constructions of mutant strains of Salmonella enterica serovar Typhi The homologous recombination,which is mediated by suicide plasmid,was used to knock out Hil D,Him A,Him D,Ydg T,Hha,HNS in S.Typhi,and the Fis and Omp R mutant strains were stored in our laboratory.2.q RT-PCR q RT-PCR was used to analyse the transcriptional level of mig-14 in S.Typhi wild type strains and mutations.Analysed the difference of mig-14 transcriptional levels in S.Typhi derivatives,which were cultured in LB medium,to demonstrate whether mig-14 transcription is repressed by HNS,Hha and Ydg T or not in a non-selected environment;Analysed the difference of mig-14 transcriptional levels in S.Typhi derivatives,which were under polymyxin B exposure,to demonstrate whether mig-14 transcription is activated by regulator Hil D,Him A,Him D,Fis,Omp R or not.3.Western blot to detect the Mig-14 level The Mig-14 antiserum prepared in previous work was used to detect Mig-14 protein levels in S.Typhi strains.4.lacZ fusion experiment The recombinant plasmid p HRP309 was constructed.Amplified the promoter region of mig-14 using PCR;Inserted the promoter region into p HRP309 plasmid,located at the upstream of the β-galactosidase gene-lac Z.The recombinant p HRP309 plasmids were obtained and transferred into strains.According to the difference of the β-galactosidase contained in strains,we can determine the mig-14 transcriptional and expression levels.5.Protein expression and purification Expressed and purificated HNS and Him A(α subunit of IHF)by E.coli BL21 to investigate the relationships between these two proteins and mig-14.6.Electrophoretic mobility shift assay The promoter region of mig-14 was co-incubated with HNS or Him A,and then subjected into electrophoresis on 6% polyacrylamide nondenaturing gels to test the binding between proteins and the promoter DNAs.7.Analysis the influences of IHF,mig-14 to iagA Constructed a him A/mig-14 double gens mutant strain of S.Typhi.Constructed the heterologous expression plasmids of him A and mig-14.Transferred the heterologous expression plasmids into him A/mig-14 double gens mutant strain respectively,analysed the iag A transcriptional level to demonstrate the regulator relationship of IHF and Mig-14 to iag A under early hyperosmotic condition.Results:1.The hil D,him A,him D,hha and ydg T mutant strains of S.Typhi were successfully constructed.Because of the failure to knock out hns gene in S.Typhi after repeated attempts,we constructed a over-expression hns strain by transferring a heterologous expression plasmids of hns into S.Typhi,and sought to demonstrate that hns could repress mig-14 transcription.2.The results of qRT-PCR indicated that the transcription of mig-14 was repressed by silencing protein-HNS/Hha/Ydg T in a non-selected environment.Both of the transcription levels of mig-14 in hha and ydg T mutants were below which in wild type strain,what’s more,the deletion of hha resulted a considerable evaluation than the deletion of ydg T did.Furthermore,over-expression of hns in S.Typhi resulted a significantly reduction of the transcription levels of mig-14.In general,the transcription of mig-14 was repressed by silencing protein-HNS/Hha/Ydg T in a non-selected environment.3.Exposed S.Typhi under polymyxin B to activate the transcription and expression of mig-14.The q RT-PCR results showed that the transcriptional levels of mig-14 were significantly reduced in fis,omp R,hil D,him A and him D mutant strains compared with the wild type strain.The results of lac Z fusion assay also indicated that the promoter activities of mig-14 were also significantly decreased in fis,omp R,hil D,him A and him D mutant strains compared with the wild type strain.4.We sought to use the Mig-14 antiserum which was prepared in previous work to test Mig-14 levels in S.Typhi strains.Unfortunately,we could not find a significantly specific bind at the corresponding site even though we checked it by mig-14 deletion mutant.So we concluded that the Mig-14 antiserum lost the specificity to Mig-14 protein.5.HNS was successfully expressed using plasmid pET-28 a,Him A was expressed utilizing p ET-15 b.Both of the proteins with 6x His Tag were purified using a Ni-colum.6.Both of HNS and Him A could bind to the promoter region of mig-14.7.him A/mig-14 double gens mutant strain of S.Typhi was successfully constructed.The results of q RT-PCR showd that the transcripition level of iag A was significantly increased after transferring a heterologous expression plasmids of him A while heterologous expression plasmids of mig-14 only could not induce the iag A.Conclusion:As a horizontal-transfer acquired gene in S.Typhi,mig-14 is repressed by silencing protein--HNS/Hha/Ydg T in a non-selected environment.When bacteria suffering from polymyxin B,the silencing of mig-14 relieves.In this study,we demonstrated that the regulators Fis,Omp R,IHF and Hil D participated in the activation of mig-14 when S.Typhi under polymyxin B exposure.HNS and Him A,as important regulators,could bound to the promoter region of mig-14.Previously,we found Mig-14 could regulate many genes located at SPI-1 which could be activated by iag A.And now we discovered that IHF could activated iag A and mig-14 in early hyperosmotic stress.More important than all of that,Mig-14 activated iag A depended on IHF.