Research On The Function Of Mig-14 Gene In Salmonella Enterica Serovar Typhi

ObjectivePrevious researches revealed that Mig-14 is an inner membrane binding protein and a putative regulator protein.Microarray analysis of global gene expression profiles during hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi) showed that the expression of mig-14 is increased at early stage of the hyperosmotic stress.The goal of the study is to clarify the function of Mig-14 on gene expression regulation at early stage of the hyperosmotic stress in S.Typhi.MethodsThe mig-14 deleted mutant ofS.Typhi was prepared by homologous recombination mediated by suicide plasmid pGMB 151.As the genomic information,two pairs of primers were designed to amplify two homologous DNA fragments(upper-and down-stream fragments of the mig-14 gene of S.Typhi) that were ligated by orientational connection as the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamH I site.The positive recombinant plasmid was selected by specific PCR and digested with BamH I.The wild-type strain of S.Typhi was then transformed with the recombinant plasmid by electroporation.The mutant mig-14~- was selected by screening on the LB sucrose plate and selected by specific PCR and finally verified by the relative DNA sequencing.The hyperosmotic stress environment was simulated by an osmotic up-shift,which increased the concentration of NaCl in the LB from 50 mM to 300 mM in vitro,the total RNA was extracted from wild-type strain and mig-14 deleted mutant of S.Typhi,respectively,at early stage of the hyperosmotic stress (30min).The gene expression profiles of wild-type and mig-14 deleted mutant of S.Typhi at early stage of the hyperosmotic stress were investigated with a genomic DNA microarray,qRT-PCR was performed for some selected genes which had significantly different expression in the mig-14 deleted mutant to prove the results of microarray.The polymyxin B sensitivity assay was performed for wild-type strain,mig-14 mutant and phoP mutant at neutral and poor acid environment respectively in order to compare their resistance to polymyxin B.ResultsPCR and sequencing analysis showed that the 390 bp in the encoding region of the mig-14 gene was deleted,and demonstrated that the mig-14 gene deleted mutant or S.enterica serovar Typhi without polar mutation was constructed successfully.Gene expression profiles analysis revealed that expression of 72 genes and 77 genes were induced and decreased,respectively,in the mig-14 mutant at early stage of hyperosmotic stress.The regulated genes were involved in flagellar proteins,invasion proteins,outer membrane proteins,metabolic enzymes and some other unkown function proteins.Expressions of genes(cydA, invF,pagP and treC) which have significantly different expression in the mig-14 deleted mutant were investigated by qRT-PCR,and the results showed that the expressions of them are consistent with the results from the microarray assay.ConclusionsThe mig-14 deleted nonpolar mutant of S.Typhi was constructed successfully.The inner membrane regulator Mig-14 of S.Typhi is important to genes expression regulation in response to the hyperosmotic stress,which involves in the assembly of flagella and the substance metabolism and the energy metabolism of S.enterica.There was nearly no difference among the sensitiveness of the three kinds of S.Typhi strain at neutral environment.However at poor acid environment,mig-14 mutant was more and less sensitive to polymyxin B than wild-type strain and phoP mutant respectively from the polymyxin B sensitivity assay.