Analysis Of Effect Of PmrA On Gene Expression Regulation Of Salmonella Enterica Serover Typhi At Later Stage Of Hyperosmotic Stress

ObjectivePmrA is a response regulator of tow-component regulatory system PmrA-PmrB,and is lowly expressed at later stage of hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi).The goal of the study is to explore the infuluence of PmrA on gene expression regulation of S. Typhi at the later stage of hyperosmotic stress.Methods(1) The pmrA deleted mutant of S.Typhi was prepared by homologous recombination mediated by suicide plasmid pGMB151.As the genomic information,two pairs of primers were designed upper-and down-stream of the pmrA gene of S.Typhi to amplify two homologous DNA fragments that were ligated by orientational connection to generate the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamH I site.The wild-type strain of S. Typhi was then transformed by the positive recombinant plasmid by electroporation.The pmrA deleted mutant was selected by screening on the 5%LB sucrose plate and verified by qRT-PCR and DNA sequencing of the pmrA gene.(2) The environmental hyperosmotic stress was simulated by an osmotic up-shift,which increased the concentration of NaCl in the LB broth from 50 mM to 300 mM in vitro.Cells were grown in low osomtic LB(50 mmol/L NaCl)(37℃,250r/min) for 4h to log-phase and then subjected to high osomtic LB(300 mmol/L NaCl) at the same conditions. The total RNA was extracted from wild-type and pmrA~- of S.Typhi at later stage of osmotic stress(120min).cDNAs were synthesized by reverse transcription and labelling with cy3- or cy5-dCTP.The gene expression profiles of wild-type strain and pmrA mutant of S.Typhi at later stage of osmotic stress were investigated by a geneomic DNA microarray analysis.(3) QRT-PCR for some genes that have significant expressional difference between in wild-type and the pmrA mutant of S.Typhi was performed to prove the results of microarray analysis.Results(1) PCR and sequencing analysis showed that the 351 bp of the pmrA gene ecoding region was deleted respectively,suggesting that the pmrA gene deleted mutant of S.Typhi was constructed successfully.(2) Gene expression profiles analysis revealed that 81 genes and 22 genes in the pmrA mutant at later stage of hyperosmotic stress were upand down-regulated,respectively.The obviously changed genes were involved in ABC transport system related proteins,flagella related proteins,invasion proteins,outer membrane proteins,enzymes and other functional proteins.(3) Result of qRT-PCR showed that the expression difference of yliC and putA in between wild-type strain and the pmrA mutant was similar with the results from the microarray assay.ConclusionsThe pmrA gene deletion muatant of S.Typhi was constructed successfully.The Regulator PmrA of S.Typhi is benificial to increase substance delivering and invasive ability by gene expression regulation in response to hyperosmotic stress.