Analysis Of Effect Of Hfq On Gene Expression Regulation Of Salmonella Enterica Serover Typhi At Earlier Stage Of Hyperosmotic Stress

ObjectiveHfq is a RNA chaperone in bacteria.The expressions of genes that involved in virulence were regulated by Hfq.In Salmonella enterica serovar Typhi(S.Typhi),many genes encoding virulence factors changed expression when S.Typhi submited hyperosmotic stress.The goal of the study is to explore the infuluence of Hfq on gene expression of S.Typhi at hyperosmotic stress.Methods1.Preparation of the hfq deleted mutant of S.Typhi.The hfq deleted mutant of S.Typhi was prepared by homologous recombination mediated.As the genomic information,two pairs of primers were designed upstream and downstream of the hfq 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 BamHI site.After cloning into E.coIi,the recombinant plasmid was validated by sequence analysis.The wild-type strain of S. Typhi was then transformed by the positive recombinant plasmid by electroporation.The recombination was visualized by PCR,and the complete recombinant strain was selected as the hfq deleted mutant strain and confirmed by the corresponding sequencing analysis.2.The gene expression profiles assay with Salmonella genomic microarray.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 rpm) for 4 h to log-phase, then NaCl was added to a final concentration of 300 mM to the low osmotic bacterial cultures,and the bacteria were incubated at 37℃with shaking for 30 min.The total RNA was extracted from wild-type and hfq deleted mutant of S.Typhi at earlier stage of osmotic stress (30 min).cDNAs were synthesized by reverse transcription and labelling with cy3- or cyS-dCTP.The gene expression profiles of wild-type strain and hfq deleted mutant of S.Typhi at earlier stage of osmotic stress were investigated by a Salmonella geneomic DNA microarray analysis.3.Supplement of the hfq gene in the hfq deleted mutant of S.Typhi. A pair of primers specific to upstream and downstream regions of the hfq gene was designed and used to amplify the hfq gene.The amplicon containing the promoter and terminater region of the hfq gene was directionally inserted into the expression vector pBAD/gⅢto form the recombinant plasmid(pBAD/hfq).The hfq deleted mutants were transformed by the positive recombinant plasmid and the empty vector to generate the hfq complementary strain and the control strain,respectively.4.Preparation of hfq-ompR double deleted mutant of S.Typhi. The ompR deleted mutant of S.Typhi was transformed by the recombinant plasmid which contains the hfq defective homologious DNA fragment by electroporation.The recombination was visualized by PCR,and the complete recombinant strain was selected as the hfq-ompR double deleted mutant strain. 5.Gene expression analysis by qRT-PCR.Primers specific to some genes that have significant expressional difference in microarray assay were designed for qRT-PCR to validate results of microarray assay.Primers specific tviA was also designed for qRT-PCR to investigate the tviA expression in wild-type,hfq mutant and hfq complementary strain under different osmolarity condition.The tviA expression was also investigated by qRT-PCR in strans of wild-type, hfq mutant and hfq-ompR double deleted mutant to determine whether the regulation of tviA expression by Hfq is associated with the OmpR-EnvZ system.Results1.PCR and sequencing analysis showed that the 132 bp of the hfq gene ecoding region was deleted,suggesting that the hfq gene deleted mutant of S.Typhi was constructed successfully.2.Gene expression profiles analysis revealed that 62 genes and 32 genes in the hfq mutant at ealier stage of hyperosmotic stress were up-regulated and down-regulated,respectively.The obviously changed genes were involved in flagella related proteins,invasion proteins,outer membrane proteins,metabolic enzymes and other functional proteins.3.PCR and relative sequencing analysis reveled that the hfq complementary strain and hfq-ompR double deleted mutant of S. Typhi were prepared successfully.4.Result of qRT-PCR showed that the expression difference of the selected gene between wild-type strain and the hfq mutant was similar with the results from the microarray assay.And the expression of tviA increased in hfq deleted mutant when S.Typhi under hyperosmotic stress but not sustained low and high osmolarity.The expression of tviA of hfq-ompR double mutant was similar with that of hfq mutant at the hyperosmotic stress.ConclusionsThe Regulator Hfq of S.Typhi is benificial to increase substance delivering and invasive ability by gene expression regulation in response to hyperosmotic stress.Expression of tviA is transiently regulated by Hfq in S.Typhi at hyperosmotic stress and the regulation is not associating with the regulator OmpR directly.