Analysis Of Effect Of UhpA On Gene Expression Regulation Of Salmonella Enterica Serover Typhi At Hyperosmotic Stress

ObjectiveUhpA is a response regulator of two-component regulatory system UhpAB,and is highly expressed at hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi).The goal of the study is to explore the infuluence of UhpA on gene expression of S.Typhi at 30 min of hyperosmotic stress.Methods1.Preparation of an uhpA deleted mutant of S.Typhi.The uhpA deleted mutant of S.Typhi was prepared by homologous recombination mediated by the suicide plasmid pGMB151.As the genomic information,two pairs of primers upper- and down-stream of the uhpA of S.Typhi were designed to amplify two homologous DNA fragments that were orientationally ligated to generate the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamHâ… site.The wild-type strain of S.Typhi was then transformed by the positive recombinant plasmid by electroporation.The uhpA deleted mutant was selected by screening on the LB with 5%sucrose plate and verified by PCR and DNA sequencing of the uhpA gene.2.Transcriptional profile assay of S.Typhi.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 mM NaCl) (37℃,250 r/min) for 4 h to log-phase,subjected to an osomtic stress (increasing NaCl to 300 mM in LB),and then incubated for 30 min. The total RNA was extracted from the wild-type and the mutant uhpA^-of S.Typhi.cDNAs were synthesized by reverse transcription and labelling with cy3- or cy5-dCTP.The gene expression profiles of wild-type strain and uhpA mutant of S.Typhi at the osmotic stress after 30min were investigated by a geneomic DNA microarray analysis.3.Quntitative real time PCR(qRT-PCR) analysis,qRT-PCR for some genes that have significant expressional difference in between wild-type and the uhpA mutant of S.Typhi was performed to prove the results of microarray analysis.4.Expression and purification of UhpA.One pair of primers was designed to amplify DNA fragments of the uhpA gene of S.Typhi, which was then inserted into the expression plasmid pET-22b. Escherichia coli JM109 was then transformed by the expression plasmid by electroporation.Expression of the UhpA protein was induced by IPTG.UhpA protein was purified with Ni-colum according the manufactory instructions.5.Gel-shifting experiment.Two pairs of primers were designed to amplify the promoter region of cysM and treB of S.Typhi.The mixtures containing 1-2μg PCR production and different amount of UhpA,GSM solution each 20μl were incubated at 30℃for 15 min befor electrophoresis.The PCR production of the eukaryotic cell was used as the negative control.The electrophoresis in 8%acrylamide gel was performed for separating PCR production.Gel was stained with Ethidium Bromide. Results1.PCR and sequencing analysis showed that the 297 bp of the uhpA ecoding region was deleted,suggesting that the uhpA gene deleted mutant of S.Typhi was constructed successfully.2.Gene expression profiles analysis revealed that 21 genes in the uhpA mutant at hyperosmotic stress after 30 min were down-regulated.The most changed genes were associated with sulfur assimilation pathways.3.Result of qRT-PCR showed that the expression difference of cysM and treB in between wild-type strain and the uhpA mutant was similar with the results from the microarray assay.4.The UhpA-his₆ protein was expressed and purified successfully for gel-shifting experiments.5.Result of gel-shifting experiments showed that UhpA could not bind poromoter regions of treB and cysM,sugesting that the expression of cysM and treB may be not regulated directly by the UhpA.ConclusionsThe uhpA gene deletion muatant of S.Typhi was constructed successfully. At up-shift high osmotic treatment,the UhpA may promoter the expression of genes involved in the metabolism of sulfur and trehalose indirectly.