Analysis Of Effect Of RpoE On Gene Expression Regulation Of Salmonella Enterica Serover Typhi In Hyperosmotic Stress

Object:Regulator RpoE is an important sigma factor which can promote lots of genes in enteroinvasive pathogen.The aim of the study is to explore the effect of the regulator RpoE respond to environment stresses and the infuluence of RpoE on gene expression regulation of Salmonella enterica serover Typhi(S.Typhi) at early stage of hyperosmotic stress.Methods:(1) The rpoE deleted mutant of S.Typhi was prepared by homologious recombination mediated by suicide plasmid.As the genomic information,two pair's primers were designed at upper- and downstreams of the rpoE gene of S.Typhi to amplify two homologous DNA fragments.A BglⅡsite was added in 5'-termini of the reverse primer of the upper stream fragment and the forward primer of the down stream fragment.After amplification and purification,two fragments were digested by BglⅡand ligated as the homologous recombinant DNA fragment,which contains the defective target rpoE gene.The homologous recombinant DNA fragment was inserted into the BamH I site of the suicide plasmid pGMB151.The positive vector was then transferred into the target cell of S.Typhi wild strain by using of electroporation.The recombination was visualized by PCR,and the complete recombinant strain was selected as the rpoE deleted mutant strain and confirmed by the corresponding sequencing analysis. (2) To compare the survival ability of wild-type strain and the rpoE mutant under the stress conditions,growth curves were drawn by using growth time as the abscissa and value of OD as the ordinate.(3) 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) at certain conditions(37℃,250r/min) for 4h to log-phase and then subjected to high osomtic LB(300 mmol/L NaCl). The total RNA was extracted from wild-type and rpoE of S.Typhi at early stage of osmotic stress(30min).cDNAs were synthesized by reverse transcription and labelling with cy3- or cy5-dCTP.The gene expression profiles of wild-type strain and rpoE mutant of S.Typhi at early stage of osmotic stress were investigated by a geneomic DNA microarray analysis.Quntitative RT-PCR for some genes that have significant expressional difference between in wild-type and the rpoE mutant of S.Typhi was performed to prove the results of microarray analysis.Results:(1) A deletion of 288bp from the rpoE gene was confirmed by PCR and sequencing analysis suggested that the rpoE deleted mutant was constructed successfully.(2) Under the conditions such as high osmolarity,acid,oxidative stress,the rpoE mutant was significantly compromised compared to the wild-type strain,but no significant different in bile stress.(3) Gene expression profiles analysis revealed that expression of 135 genes varied obviously in the rpoE mutant at early stage of hyperosmotic stress,which was involved in heat shock protein,flagella related proteins, invasion protein,outer membrane protein,enzymes,two-Component Regulatory System PhoP-PhoQ and a lot of hypothetical proteins.Conclusions:The muatant rpoE of S.Typhi was constructed successfully and the rpoE mutant was significantly compromised compared to the wild-type strain in high osmolarity,acid stress,oxidative stress.The regulator RpoE of S.Typhi was important in genes expression regulation in response to hyperosmotic stress.